Platelet-derived growth factor-responsive neural precursor cells and progeny thereof

ABSTRACT

This invention provides platelet-derived growth factor-responsive neural precursor (PRP) cells and methods of producing such cells in vivo or in vitro. These cells can further be used to generate neurons, oligodendrocytes and/or astrocytes.

RELATED APPLICATIONS

This application claims priority to application Ser. No. 60/632,751,filed Dec. 1, 2004, and application Ser. No., filed Nov. 21, 2005, eachof which are expressly incorporated herein by reference.

INTRODUCTION

Although there is general agreement about the factors involved in thedevelopment of oligodendrocyte progenitors (OLPs) throughout the centralnervous system, their precise phenotype potential is highly contentious(Liu et al., Trends Neurosci 26:410 (2003); Noble et al., Dev. Bio265:33 (2004); Rowitch, Nat Rev Neurosci 5:409 (2004)). Findings in thespinal cord, which show that similar levels of sonic hedgehog (SHH)signaling can induce motor neuron and oligodendrocyte cell fates(Pringle et al., Dev Biol 177:30 (1996); Orentas et al., Development126:2419 (1999)) and that the basic helix-loop-helix transcriptionalrepressor Olig2 is required for the generation of both cell types (Lu etal., Cell 109:75 (2002)), are consistent with the idea thatoligodendrocytes and motor neurons are generated by a common progenitor.In the brain, crosses of Olig1-CRE and Rosa-lox-β-Gal mice have revealedmutually exclusive expression of β-Gal and the astrocyte antigen S100β,while neurons and oligodendrocytes were labeled with β-Gal (Lu et al.,Cell 109:75 (2002)) suggesting the latter were generated by a commonprecursor. However, the broad expression of OLIG1/2 in the embryonicforebrain compared to platelet-derived growth factor receptor-α (PDGFRα)(Tekki-Kessaris et al., Development 128:2545 (2001)), an early OLPantigen, makes it difficult to determine whether neurons are generatedby embryonic OLPs or non-related OLIG-expressing progenitors. Furthersupport for a forebrain neuron/oligodendrocyte progenitor comes fromobservations that the tangential migration of both γ-aminobutyric acid(GABA)ergic interneurons and oligodendrocytes is disrupted in Dlx1/2null mice (Yung et al., Proc Natl Acad Sci USA 99:16273 (2002)).However, the fact that taulacZ-positive astrocytes appear in mice thatexpress taulacZ under the Dlx2 promoter (Marshall et al., J Neurosci22:9821 (2002)) suggests that DLX-expressing cells may be either bemultipotent or that DLX-expressing progenitors are a heterogeneouspopulation.

In contrast to the studies reported above, crosses of Olig1-CRE andRosa-lox-β-Gal mice have also shown that some OLIG1-expressing cells inthe spinal cord eventually become astrocytes (Liu et al., Glia 45:67(2004)), thereby providing in vivo evidence for anoligodendrocyte/astrocyte progenitor. Furthermore, the in vitroisolation of glial-restricted precursors (GRPs) from the spinal cord(Rao et al., Dev Biol 188:48 (1997); Rao et al., Proc Natl Acad Sci USA95:3996 (1998)), and their transplantation and differentiation intoastrocytes and oligodendrocytes (Rao et al., Dev Biol 188:48 (1997); Raoet al., Proc Natl Acad Sci USA 95:3996 (1998); Herrera et al., ExpNeurol 171:11 (2001)), supports such a lineage model. However, the factthat GRPs can be isolated from dorsal as well as ventral embryonicspinal cords contrasts with studies demonstrating the ventralrestriction of OLPs (Warf et al., J Neurosci 11:2477 (1991); Pringle etal., Development 117:525 (1993); Ono et al., Development 121:1743(1995); Lu et al., Cell 109:75 (2002); Zhou et al., Cell 109:61 (2002)).This may be reconciled by the findings of Gabay et al., Neuron 40:485(2003), who found that the deregulation of dorsoventral patterning invitro, due in part to aberrant SHH production induced by FGF signaling,may be responsible for the generation of oligodendrocytes bydorsally-derived GRPs. Nevertheless, a variety of studies in the brain,including in vivo retroviral-mediated lineage studies of the ratpostnatal cerebral cortex (Levison et al., Development 119:611 (1993);Levison et al., Neuron 10:201 (1993)) as well as in vitrocharacterization of cortical OLPs (Mabie et al., J Neurosci 17:4112(1997)) and optic nerve O-2A progenitors (Temple et al., Nature 313:223(1985)), which never generate neurons, unless they are reprogrammed to,become NSCs by their differentiation into astrocytes and subsequentexpansion in FGF2 (Kondo et al., Science 289:1754 (2000)), support thecontention that astrocytes and oligodendrocytes are generated by acommon progenitor. However, retroviral tracing of the prenatal ratcortex revealed that glial clones were either oligodendroglial orastroglial (Parnavelas, Exp Neurol 156:418 (1999)), although the samestudy also found mixed oligodendrocyte and astrocyte clones wheninjections of retrovirus were made into the postnatal SVZ.Interestingly, progenitors that express NG2, a chondroitin proteoglycanpreviously shown to co-localize to O-2A progenitors in vivo (Nishiyamaet al., J Neurosci Res 43:299 (1996)), have been found to generateneurons in the postnatal hippocampus (Belachew et al., J Cell Biol161:169 (2003)). However, these progenitors express the EGF receptor,have been identified as transit-amplifying type C-like mulitpotent cells(Aguirre et al., J Cell Biol 165:575 (2004)), and therefore whether theyrepresent the differentiation properties of an OLP population isquestionable. Thus, the cell types OLPs produce in the developingembryonic forebrain remains unclear.

SUMMARY

PDGF-responsive neural precursor (PRP) generated clonal cell expansionscan be obtained from the medial ganglionic eminence (MGE), and PRPprogeny can differentiate into parvalbumin-positive interneurons,oligodendrocytes, and astrocytes. Thyroid hormone (e.g., T3) and bonemorphogenetic protein-2 (BMP-2) promote a mutually exclusivedifferentiation of oligodendrocytes and neurons, respectively, whileciliary neurotrophic factor (CNTF) acts with BMP-2 to suppress OLIG-2expression and promote astroglial differentiation from PRP cells. PRPsclonally proliferate or undergo self-renewal in the presence offibroblast growth factor-2 (FGF-2) with PDGF, which is dependent uponsonic hedgehog signaling (SHH). Evidence that forebrain oligodendrocytesand parvalbumin-positive interneurons are generated by a commonprecursor cell (PRP), and the signals regulating the multipledifferentiation routes of PRP precursor cell progeny, is disclosedherein.

Isolated and purified mammalian platelet derived growth factor(PDGF)-responsive neural precursor (PRP) cells are provided, optionallyexpressing PDGF receptor alpha. In one embodiment, a cell, whencontacted with one or more of thyroid hormone, bone morphogeneticprotein-2 (BMP-2), ciliary neurotrophic factor (CNTF) ortriiodothyronine (T3), gives rise to a differentiated neural cell thatexpresses detectable amounts of one or more protein markers selectedfrom: GABA, parvalbumin, beta-II tubulin, calbindin D, calretinin, O4,neurofilament M (NFM), myelin basic protein (MBP), TOA-64/TUC-2 andGFAP. In another embodiment, a cell, when contacted with one or more ofthyroid hormone, BMP-2, CNTF or T3, gives rise to a differentiatedneuron, oligodendrocyte, astrocyte or mixture thereof.

Isolated and purified mammalian platelet derived growth factor(PDGF)-responsive neural precursor (PRP) cells are provided, optionallyexpressing PDGF receptor alpha, in which cells exhibit greater or lessclonal proliferation when contacted with a factor or stimuli, orsubjected to a condition, in vitro or in vivo. In various embodiments, acell exhibits greater clonal proliferation when contacted with a PDGFreceptor (PDGFR) agonist and an fibroblast growth factor (FGF) receptoragonist, then when contacted with either PDGF alone or epidermal growthfactor (EGF) alone; a cell exhibits greater clonal proliferation whencontacted with PDGF and brain derived neurotrophic factor (BDNF), thenwhen contacted with either PDGF alone or EGF alone; or a cell exhibitsgreater clonal proliferation when contacted with PDGF and NT-3, thenwhen contacted with either PDGF alone or EGF alone. In various aspects,clonal proliferation is induced or increased by stimulating sonichedgehog signaling (SHH), or clonal proliferation is reduced orprevented by inhibition of sonic hedgehog signaling (SHH). In variousadditional embodiments, a cell exhibits less clonal proliferation underconditions of contact with PDGF than clonal proliferation of neural stemcell (NSC) under conditions of contact of NSC with EGF, a cell does notproliferate when contacted with EGF alone or FGF2 alone, or a cellproliferates when contacted with a PDGF receptor agonist and an FGFreceptor agonist. Ina further embodiment, a cell exhibits increasedclonal proliferation when contacted with a PDGF receptor agonist and anFGF receptor agonist, as compared to clonal proliferation when contactedwith PDGF alone, EGF alone or FGF2 alone.

Cells developmentally intermediate in the lineage with respect to PRPcells are and progeny thereof are also provided. In one embodiment, anintermediate cell is intermediate with respect to an undifferentiatedcell and a neuron or oligodendrocyte. In another embodiment, anintermediate cell is designated an N/O cell and gives rise to adifferentiated neuron or oligodendrocyte, but not an astrocyte, whencontacted with one or more of BMP-2 or T3.

Isolated and purified populations of mammalian platelet derived growthfactor (PDGF)-responsive neural precursor (PRP) cells, optionallyexpressing PDGF receptor alpha are provided, including progeny thereof.In one embodiment, at least a portion of the cell population gives riseto a differentiated neuron when contacted with BMP-2 and into anoligodendrocyte when contacted with triiodothyronine (T3). In anotherembodiment, at least a portion of the cell population gives rise to adifferentiated astrocyte when contacted with BMP-2 and CNTF. In afurther embodiment, at least a portion of the cell population gives riseto a differentiated astrocyte when contacted with T3 followed by contactwith BMP-2 and CNTF. In an additional embodiment, the cells do not giverise to substantial numbers or detectable differentiated astrocytes, bycontact with BMP-2 alone or CNTF alone.

Isolated and purified mammalian cell culture comprising undifferentiatedand differentiated neural cells, optionally expressing PDGF receptoralpha, are further provided, including progeny thereof. In oneembodiment, a cell culture includes about ⅓ of the total number of cellscomprise differentiated beta-III-tubulin expressing neurons, anddifferentiated astrocytes are fewer in number or absent; or about ⅓ ofthe total number of cells comprise differentiated beta-III-tubulinexpressing neurons and about ⅓ of the total number of cells in theculture comprise differentiated oligodendrocytes, and differentiatedastrocytes are fewer in number or absent; or differentiated astrocytesare present in the cell culture, and ⅓ or less of the total number ofcells in the culture comprise differentiated neurons; or about ⅔ of thetotal number of cells in the culture comprise differentiated astrocytes,and ⅓ or less of the total number of cells in the culture comprisedifferentiated neurons. In another embodiment, a cell culture includesneurons, and optionally at least 50%, 60%, 70%, 80% or more of theneurons express detectable amounts of parvalbumin or GABA.

Isolated and purified mammalian PRP cells, optionally expressing PDGFreceptor alpha substantially free of connective tissue, are additionallyprovided, including progeny thereof. Isolated and purified PRP cells,optionally expressing PDGF receptor alpha dissociated from other cellsor tissue, are additionally provided, including progeny thereof. In oneaspect, PRP cell or progeny thereof have been contacted with a PDGFRagonist. In another aspect, PRP cell or progeny thereof are a culture ofcells substantially free of differentiated neural cells.

Isolated and purified mammalian PRP cells, optionally expressing PDGFreceptor alpha substantially including progeny thereof, include cellsdistinct from EGF-responsive neural stem cell (NSC). In one embodiment,a cell is more motile as compared to a progeny of EGF-responsive neuralstem cell (NSC).

Mammalian PRP cells and progeny thereof can be obtained or derived froma nerve tissue or organ. In one embodiment, a cell includes or isderived from a primary brain cell isolate. In another embodiment, a cellincludes or is derived from ganglionic eminence (e.g., medial ganglioniceminence, MGE).

Isolated and purified populations of mammalian platelet derived growthfactor (PDGF)-responsive neural precursor (PRP) cells, optionallyexpressing PDGF receptor alpha are provided, including progeny thereofand cell populations, that have been contacted with a factor or stimuli,or subjected to or exposed to a condition, in vitro or in vivo. In oneembodiment, a cell is or has been contacted with one or more of: PDGF,BDNF, NT-3, thyroid hormone, BMP-2, CNTF, EGF and T3.

Mammalian PRP cells and progeny thereof include human, primate, murine,rattus, bovine, porcine, equine, avian, cavia, lagomorph, canine orfeline cells. Mammalian PDGF-responsive neural precursor (PRP) cellsinclude cells obtained or derived from mammals; from an embryo, fetus,juvenile or adult.

Mammalian PRP cells and progeny thereof transformed with a nucleic acidare further provided. In one embodiment, a nucleic acid encodes aprotein. In various aspects, a protein is a neurotransmitter,neurotransmitter receptor, growth factor, growth factor receptor,neurotransmitter-synthesizing enzyme, neurotransmitterreceptor-synthesizing enzyme, growth factor-synthesizing enzyme, growthfactor receptor-synthesizing enzyme, or a neuropeptide. In particularaspects, a protein is selected from brain-derived neurotrophic factor,neurotrophin, CNTF, amphiregulin, basic FGF, acidic FGF, EGF,transforming growth factor-alpha, transforming growth factor-beta, PDGF,insulin-like growth factor and interleukin. In additional particularaspects, a protein is selected from a low affinity nerve growth factorreceptor, CNTF receptor, neurotrophin receptor, EGF receptor, FGFreceptor and amphiregulin receptor. In further particular aspects, aprotein is selected from a substance-P, neuropeptide-Y, enkephalin,vasopressin, vasoactive intestinal peptide, cholecystokinin, glucagon,bombesin, somatostatin, tachykinin, endorphin and calcitoningene-related peptide. In still further particular aspects, a protein isselected from a tyrosine hydroxylase, tryptophan hydroxylase,phenylethanolamine N-methyltransferase, histidine decarboxylase,glutamic acid decarboxylase, choline acetyltransferase, dopadecarboxylase, dopamine beta hydroxylase and amino acid decarboxylase.

Cell cultures including PRP cell that express PDGF receptor alpha,including progeny thereof and cell populations, that have been contactedwith a factor or stimuli, or subjected to or exposed to a condition, invitro or in vivo, are additionally provided. In one embodiment, a cellculture has been contacted with a thyroid hormone, BMP-2, CNTF or T3,which gives rise to a differentiated neural cell that expressesdetectable amounts of one or more protein markers selected from: GABA,parvalbumin, beta-II tubulin, calbindin D, calretinin, O4, neurofilamentM (NFM), myelin basic protein (MBP), TOA-64/TUC-2 and GFAP. In anotherembodiment, a cell culture has been contacted with one or more ofthyroid hormone, BMP-2, CNTF or T3, which gives rise to a differentiatedneural cell that expresses detectable amounts of one or more proteinmarkers selected from: GABA, parvalbumin, beta-II tubulin, calbindin D,calretinin, O4, neurofilament M (NFM), myelin basic protein (MBP),TOA-64/TUC-2 and GFAP.

Cell cultures including populations of cells enriched for mammalianPDGF-responsive neural. precursor (PRP) cells that optionally expressPDGF receptor alpha are moreover provided, In one embodiment, at least aportion of the enriched cells, when contacted with one or more ofthyroid hormone, BMP-2, CNTF or T3, gives rise to a differentiatedneural cell that expresses detectable amounts of one or more proteinmarkers selected from: GABA, parvalbumin, beta-II tubulin, calbindin D,calretinin, O4, neurofilament M (NFM), myelin basic protein (MBP),TOA-64/TUC-2 and GFAP. In another embodiment, at least a portion of theenriched cells, when contacted with one or more of thyroid hormone,BMP-2, CNTF or T3, gives rise to a differentiated neuron,oligodendrocyte, astrocyte or mixture thereof.

Progeny of PDGF-responsive neural precursor (PRP) cells are provided.Progeny include clonally expanded cells, progenitor cells, anddifferentiated cells. Progeny include first, second, third, fourth,fifth, sixth seventh or any subsequent generation progeny cell or cells.

Pharmaceutical compositions including mammalian PDGF-responsive neuralprecursor (PRP) cells, as well as clonally expanded, progenitor ordifferentiated progeny cells thereof, and a pharmaceutically acceptablecarrier or excipient, are provided. Kits including mammalianPDGF-responsive neural precursor (PRP) cells, as well as clonallyexpanded, progenitor or differentiated progeny cells thereof, andpharmaceutical compositions are also provided.

Methods of producing mammalian PDGF-responsive neural precursor (PRP)cells that optionally express PDGF receptor alpha, in vitro and in vivo,are provided. In one embodiment, a method includes culturing brainmedial ganglionic eminence in a culture medium containing PDGF underconditions allowing clonal proliferation or differentiation of the PRPcells. In various aspects, a culture medium or administration does notinclude EGF or FGF2; a culture medium contains one or more of PDGF,thyroid hormone, BMP-2, CNTF, T3, PDGF, BDNF, NT-3 or FGF2.

In another embodiment, a method includes administering a PDGFR agonistto the mammal in an effective amount for delivery of the PDGFR agonist(e.g., PDGF) to increase PRP cell numbers. In one aspect, a mammal doesnot receive EGF or FGF. In additional aspects, a mammal is administeredFGF2, BDNF or NT-3 substantially simultaneously with the PDGFR agonist.In another aspect, PDGFR agonist is administered locally, regionally orsystemically, for example, to the brain (cranium) of the mammal. Invarious additional aspects, administration occurs intracranially,intravenously, intravascularly, intramuscularly, subcutaneously,intraperitoneally, topically, orally, nasally or by inhalation.

Methods of increasing oligodendrocytes, neurons or astrocytes in amammal are also provided. In one embodiment, a method includesadministering an effective amount of PDGFR agonist to the mammal toproliferate PRP cells; and administering an effective amount of thyroidhormone or T3 to increase oligodendrocytes, BMP-2 to increase neurons,or both BMP-2 and CNTF to increase astrocytes. In various aspects, FGF2,BDNF or NT-3 is administered substantially simultaneously with the PDGFRagonist to the mammal.

In a further embodiment, a method of producing oligodendrocytes includesculturing brain tissue from a mammal in a culture medium comprising aPDGFR agonist and allowing proliferation of PRP cells; anddifferentiating the proliferated PRP cells to produce oligodendrocytes,for example, by contacting the proliferated PRP cells with an effectiveamount of thyroid hormone or T3. In one aspect, the oligodendrocytes arecontacted with an effective amount of BMP-2 and CNTF to produce neuronsand astrocytes.

In a another embodiment, a method of producing neurons includesculturing brain tissue from a mammal in a culture medium comprisingPDGFR agonist and allowing proliferation of PRP cells; anddifferentiating the proliferated PRP cells to produce neurons, forexample, by contacting the proliferated PRP cells with an effectiveamount of BMP-2.

In an additional embodiment, a method of producing astrocytes, includesculturing brain tissue from a mammal in a culture medium comprisingPDGFR agonist and allowing proliferation of PRP cells; anddifferentiating the proliferated PRP cells to produce astrocytes, forexample, by contacting the proliferated PRP cells with an effectiveamount of BMP-2 and CNTF.

Methods of the invention include clonally expanding PRP cells. Forexample, PRP cells may be clonally expanded by contacting PRP cells withPDGF and FGF-2; or PDGF and BDNF; or PDGF and NT-3 prior todifferentiating cells.

Administration in accordance with the invention includes intracranial,intravenous, intravascular, intramuscular, subcutaneous,intraperitoneal, topical, oral, nasal and inhalation. Mammals targetedfor administration or in vivo delivery include humans, primates, murine,rattus, bovine, porcine, equine, avies, cavias, lagomorphs, canines andfelines. Mammals include, for example, subjects in need of increasednumbers of PRP cells, progenitor cells, oligodendrocytes, neurons orastrocytes, or progeny thereof. Mammals further include, for example,subjects suffering from a loss of or injury to oligodendrocytes, neuronsor astrocytes; subjects' afflicted with or at risk of affliction with aneurological disease or disorder (e.g., affects central nerves, such asbrain or spinal cord, or affects peripheral nerves, such as motor,sensory or autonomic nerves), or undesirable medical condition.Exemplary neurological diseases and undesirable medical conditionsinclude neurodegenerative diseases, stroke (e.g., hemorrhagic stroke,focal ischemic stroke or global ischemic stroke), aneurysm, brain orspinal cord injury or cranium or spinal column trauma. Brain or spinalcord injury, or cranium or spinal column trauma, can be caused by astroke or surgery.

Compositions and methods of the invention include inducing clonalproliferation or self-renewal of the PRP cells. In one embodiment,clonal proliferation or self-renewal is induced by contacting the PRPcells with PDGF and FGF-2; or PDGF and BDNF; or PDGF and NT-3. Inanother embodiment, a majority of the clonally proliferated cells arenot differentiated into neurons, oligodendrocytes or astrocytes. In afurther embodiment, a majority of the differentiated cells are neurons,oligodendrocytes, astrocytes or a combination thereof. Clonally expandedor self-renewed population of cells produced by the various methods aretherefore also provided.

Methods for treating or ameliorating a disease, disorder or undesirablemedical condition associated with neuron, oligodendrocytes or astrocyteloss, injury or dysfunction are provided. In one embodiment, a methodincludes transplanting an effective amount of the PRP cells or progenythereof, to a mammal harboring the disease, disorder or medicalcondition. In another embodiment, a method includes administering aneffective amount of PDGFR agonist to a mammal harboring the disease,disorder or medical condition, as well as one or more of FGF-2, thyroidhormone, T3, BMP-2 or CNTF. Methods of treatment additionally includeembodiments that include administering one or more agents selected fromPDGF; PDGF and FGF-2; PDGF and BDNF; PDGF and NT-3; thyroid hormone; T3;BMP-2; BMP-2 and CNTF.

Methods of treatment include treating a neurological injury or trauma,for example, which affects central or peripheral nerves (e.g., affectscentral nerves, such as brain or spinal cord, or affects peripheralnerves, such as motor, sensory or autonomic nerves). Exemplaryneurological diseases and undesirable medical conditions includeneurodegenerative diseases, stroke (e.g., hemorrhagic stroke, focalischemic stroke or global ischemic stroke), aneurysm, brain or spinalcord injury or cranium or spinal column trauma. Brain or spinal cordinjury, or cranium or spinal column trauma, can be caused by a stroke orsurgery. Exemplary neurological diseases and undesirable medicalconditions further include Alzheimer's Disease, multiple sclerosis (MS),macular degeneration, glaucoma, diabetic retinopathy, peripheralneuropathy, Huntington's Disease, amyotrophic lateral sclerosis (ALS),Parkinson's Disease, stroke, depression, epilepsy, neurosis andpsychosis.

Methods of identifying agents that modulate clonal proliferation or selfrenewal or differentiation of a neural precursor cell are provided. Inone embodiment, a method includes providing the PRP cells or progenycells thereof; contacting the cells of step (a) with a candidate agent;and determining if the candidate agent modulates clonal expansion ordifferentiation of the cells. In one aspect, formation of progeny (e.g.,neurospheres) is determined. In another aspect, differentiation into oneor more of neurons, oligodendrocytes or astrocytes is determined.

DESCRIPTION OF DRAWINGS

FIGS. 1A-1G show data indicating that PDGF induces proliferation ofprecursors from the MGE that can differentiate into neurons andoligodendrocytes. A, PDGF-AA induces generation of neurospheres in adose-dependent manner. B, Significantly more neurospheres were generatedfrom the MGE than the LGE by either PDGF-AA or PDGF-BB. C, D, GFP- andnon-GFP-expressing dissociated E14 MGEs generated neurospheres notchimeric for GFP-expression, indicating clonal proliferation of PRPs. E,PDGF-generated neurospheres differentiated into oligodendrocytes and F,parvalbumin-immunoreactive GABA-ergic interneurons. G, Photomicrographsof PDGFRα-expressing precursor cells that co-express neuron-specificantigen TOAD-64 within the E14 forebrain. Scale bars for C,E,F,G are 50,50, 25, and 25 μm, respectively.

FIGS. 2A-2H show data indicating that PRPs are distinct fromEGF-responsive NSCs. A, Low power photomicrograph illustrating PDGFRαwithin the AEP (inset), as well as in the primordium of the choroidplexus (arrow). High power photomicrographs of B, PDGFRα- and C, EGFreceptor-expressing separate precursor populations (merged, D). E,Greater numbers of neurospheres generated in PDGF-AA and EGF compared toPDGF-AA or EGF alone. F, PRPs have a limited self-renewal capacity whensingle neurospheres of the same size were passaged in PDGF or EGFcompared to EGF-generated neurospheres passaged in PDGF. G,PDGF-generated differentiated progeny migrated large distances away fromthe center of differentiating neurospheres in comparison to H,EGF-generated neurospheres, which were rarely seen migrating away fromneurospheres. Scale bars for A, inset in A, D, and H are 200, 50, 50,and 100 μm, respectively. Asterisks in B and C indicate autofluorescentblood cells.

FIGS. 3A-3F show data indicating that BMP-2 and T3 promotedifferentiation of neurons and oligodendrocytes from PDGF-generatedneurospheres, respectively. Primary PDGF-AA-generated neurospheresdifferentiated for 2 DIV in A, 1% FBS; B, BMP-2; C, T3; or D, T3 andBMP-2 analyzed for immunocytochemistry against β-III-tubulin (neurons),O4 (oligodendrocytes), and Hoechst (nuclei, blue); and E, numbers ofimmunoreactive cells. F, cells in 1% FBS with an oligodendroglialmorphology express both O4 and β-III-tubulin. Scale bars in D and F are50 μm and 12.5 μm, respectively.

FIGS. 4A-4C show data indicating that T3 promotes and BMP-2 inhibitsexpression of mature oligodendroglial antigens in differentiating,primary PDGF-generated neurospheres. A, Photomicrograph of MBP- andNFM-immunoreactivity and Hoechst nuclear staining in PDGF-generatedneurospheres differentiated in 1% FBS. B, BMP-2 increased percentage ofclones expressing NFM compared to 1% FBS. T3 promoted oligodendrocytematuration. Cells immunoreactive for both NFM and MBP were not observed.C, T3 increased MBP-expressing cell numbers compared to 1% FBS andBMP-2, which was suppressed by BMP-2. Scale bar=12.5 μm.

FIGS. 5A-5F show data indicating that BMP-2 and CNTF promote astroglialdifferentiation of an apparently distinct cell population. A, Astroglialdifferentiation of PDGF-generated neurospheres is evident after 2 daystreatment with BMP-2 and CNTF. B, neuronal differentiation ofPDGF-generated neurospheres is not suppressed by BMP-2 and CNTFtreatment. C, differentiation in T3 promoted oligodendroglial and notastroglial differentiation in primary PDGF-generated neurospheres, andD, addition of BMP-2 and CNTF after the second day resulted in greatercell survival and a significant number of cells adopted an astroglialcell fate, but not at the expense of oligodendrocytes. E, F, BMP-2suppresses O4 expression as efficaciously as BMP-2 and CNTF together,but BMP-2 and CNTF together are more effective at suppressing OLIG2expression than BMP-2 alone. Scale bars in A, B, C, inset in C, and Eare 50, 50, 50, 25, and 25 μm, respectively. TC=total cell number.

FIGS. 6A-6D show data indicating that SHH signaling promotes generationof primary neurospheres by PRPs. A, PDGF-generated neurospheres, inDMSO. B, cyclopamine reduces the size and numbers of PDGF-generated.Arrows illustrate normally differentiating cells, indicating the effectof cyclopamine is not due to toxicity. D, SHH signaling significantlyenhances the generation or primary PDGF-generated neurospheres incomparison to PDGF alone. Scale bar in B is 100 μm.

FIGS. 7A-7G show data indicating that PDGF and FGF2 signaling promoteself-renewal of PRPs through an SHH-dependent pathway. GFP-expressingPDGF-generated neurospheres in PDGF A, without, or B, with EGF-feederlayer. C, numbers in parenthesis indicate number of neurospheresexamined. D, Photomicrograph of GFP-expressing cells within a clone thatexpress PDGFRα (indicated by arrows). E, no significant increase ingeneration of secondary neurospheres when primary PRPs were grown inconjunction with SH or when passaged into PDGF and SHH, compared toneurospheres generated and passaged in PDGF alone. F, PDGFRα-expressingcells co-express FGFR2 in the E14 MGE. G, FGF2 by itself had nosignificant effect on generation of secondary neurospheres byPDGF-generated neurospheres, except when combined with PDGF. SHHpromoted generation of secondary neurospheres in FGF2, but not asrobustly as PDGF and FGF2. Scale bars in B, inset in B, D, inset in D,and F are 100, 50, 50, 25, and 50 μm, respectively.

FIG. 8 is a schematic representation of self-renewal and differentiationof E14 ventral forebrain PRPs. NSCs generate PRPs in both PDGF and FGFto activate the SHH pathway for continued expansion. After expansion,levels of OLIG expression as well as the environment determine the fateof PRPs. High levels of OLIG expression, maintained by T3, supportoligodendroglial differentiation, whereas decreasing levels of OLIG2expression in the presence of BMP or BMP and CNTF promote generation ofneurons and astrocytes, respectively.

FIGS. 9A-9C show that neurospheres are produced when PRPs are generatedin the presence of PDGF and BDNF or NT-3, but not NGF.

FIG. 10 shows that NT-3 and BDNF promote generation of largerneurospheres in the presence of PDGF.

FIG. 11 shows that PRPs co-express PDGFRα and TrkC in the E14 ventralforebrain.

FIG. 12 shows that PRPs do not co-express PDGFRα and TrkB in the E14ventral forebrain.

FIG. 13 shows that NT-3 apparently does not maintain the PRP populationby promoting cell survival.

FIG. 14 shows that an initial 24 hour treatment with NT-3 was moreeffective at promoting generation of neurospheres than continuedexposure to NT-3 after the first 24 hours.

FIG. 15 shows that neurospheres initially generated in PDGF and NT-3produced more secondary neurospheres in either condition.

DETAILED DESCRIPTION

The invention provides clonally-derived, self-renewing PRP cells. Theinvention also provides PRP progeny cells, including clonally-derived,self-renewing cells, progenitor cells, and differentiated cells. PRPcells have the capacity to generate neurons, oligodendrocytes, andastrocytes. PRP cells are distinct from cells generated by epidermalgrowth factor (EGF)-responsive neural stem cells (NSCs) in severalrespects. When differentiated in FBS, PRP progeny cells differentiateinto neurons and oligodendrocytes, whereas EGF-generated progenydifferentiate into neurons, oligodendrocytes, and astrocytes. PRPs arenot self-renewing when passaged in EGF, whereas EGF NSCs are. Progenycells of PRPs are highly motile in comparison to EGF-generated progeny.

PRPs are a neural precursor cell capable of forming progenitor cells orneurons and both types of macroglia during forebrain development. PRPsexhibit a limited capcity for self-renewal under conditions of passagewith PDGF, which can be enhanced by fibroblast growth factor-2 (FGF2), aprocess dependent at least in part upon SHH. PRP cells may have anunlimited capcity for self-renewal when passged with other factors orstimuli or under different conditions. PRP undergoes a series ofsymmetric and asymmetric cell divisions, to produce more of itself(self-renewal/clonal expansion) and a cell with the potential todifferentiate into either a neuron or oligodendrocyte (N/O cell). InBMP-2 and CNTF, the majority of undifferentiated PRPs differentiate intoastrocytes, which reduces the number of undifferentiated cells withoutaffecting neuron numbers induced by BMP-2's action on the N/O cell.Astrocyte generation appears to be direct from PRPs and separate fromthe N/O cell because clones containing both astrocytes andoligodendrocytes are observed when PDGF-generated neurospheres aredifferentiated in triiodothyronine (T3) followed by the addition ofBMP-2 and CNTF for the remainder of the differentiation period.

PRPs are a unique population of oligodendrocyte precursors, with bothdistinct and similar properties to other OLPs described previously (Liuet al., Trends Neurosci 26:410 (2003); Noble et al., Dev Bio 265:33(2004); Rowitch, Nat Rev Neurosci 5:409 (2004)). PRPs are heterogeneousin their ability to generate neurons and subtypes of astrocytes. Thedevelopment of OLPs suggests that OLPs in vivo are also a heterogeneouspopulation. Even within the forebrain, based on the expression ofTOAD-64 in PRPs, there appears to be heterogeneity. PRPs may maintainthe capacity to generate neurons through to adulthood. Human PRPsgenerated as neurospheres permit isolating and expanding neuralprecursors or differentiated progeny for transplantation in white matterfor the treatment of central nervous system (CNS) or peripheral nervoussystem (PNS) trauma, injury, a disease or disorder, or undesirablemedical condition.

In accordance with the invention, there are provided isolated andpurified mammalian platelet derived growth factor (PDGF)-responsiveneural precursor (PRP) cells, wherein said cells express PDGF receptoralpha. In one embodiment, a cell, when contacted with one or more ofthyroid hormone, bone morphogenetic protein-2 (BMP-2), ciliaryneurotrophic factor (CNTF) or triiodothyronine (T3), gives rise to adifferentiated neural cell that expresses detectable amounts of one ormore protein markers selected from: GABA, parvalbumin, beta-II tubulin,calbindin D, calretinin, O4, neurofilament M (NFM), myelin basic protein(MBP), TOA-64/TUC-2 and GFAP. In another embodiment, a cell, whencontacted with one or more of thyroid hormone, BMP-2, CNTF or T3, givesrise to a differentiated neuron, oligodendrocyte, astrocyte or mixturethereof. In an additional embodiment, at least a portion of cells giverise to a differentiated a neuron when contacted with BMP-2 and givesrise to a differentiated oligodendrocyte when contacted withtriiodothyronine (T3). In a particular aspect, at least a portion ofcells give rise to differentiated astrocytes when contacted with BMP-2and CNTF. In another particular aspect, at least a portion of cells giverise to differentiated astrocytes when contacted with T3 followed bycontact with BMP-2 and CNTF. In a further particular aspect, cells donot give rise to differentiated astrocytes by contact with BMP-2 aloneor CNTF alone.

Further provided are cell intermediates that are progeny of anundifferentiated cell (e.g., PRP), but are not fully lineage committedor differentiated. In one embodiment, a cell is intermediate withrespect to the mammalian PDGF-responsive neural precursor (PRP) cell anda neuron or oligodendrocyte, and the intermediate cell is designated anN/O cell, which can give rise to differentiated neurons oroligodendrocytes, but not astrocytes, when contacted with one or more ofBMP-2 or T3.

In accordance with the invention, also provided are isolated andpurified mammalian PDGF-responsive neural precursor (PRP) cell, whereinthe cell expresses PDGF receptor alpha. In one embodiment, a cellexhibits greater clonal proliferation when contacted with a PDGFreceptor (PDGFR) agonist and a fibroblast growth factor (FGF) receptoragonist, then when contacted with either PDGF alone or epidermal growthfactor (EGF) alone. In another embodiment, a cell exhibits greaterclonal proliferation when contacted with PDGF and brain derivedneurotrophic factor (BDNF), then when contacted with either PDGF aloneor EGF alone. In a further embodiment, a cell exhibits greater clonalproliferation under conditions of contact with PDGF and NT-3, then underconditions of contact with either PDGF alone or EGF alone. In anadditional embodiment, a cell exhibits less clonal proliferation underconditions of contact with PDGF than clonal proliferation of neural stemcell (NSC) under conditions of NSC contact with EGF. Under differentconditions, clonal proliferation may be different between PRP and NSCcells. In still further embodiments, a cell 1) does not form aneurosphere when contacted with PDGF alone, EGF alone or FGF2 alone; orforms a neurosphere when contacted with a PDGF receptor agonist and anFGF receptor agonist; or exhibits increased clonal proliferatation whencontacted with a PDGF receptor agonist and an FGF receptor agonist, ascompared to clonal proliferation when contacted with PDGF alone, EGFalone or FGF2 alone. In various aspects of the embodiments set forthherein, clonal proliferation is induced or increased by stimulatingsonic hedgehog signaling (SHH), and clonal proliferation is reduced orprevented by inhibition of sonic hedgehog signaling (SHH).

Additionally provided are cell cultures including undifferentiated anddifferentiated neural cells in varying proportions or cell numbers. Inone embodiment, about ⅓ of the total number of cells in the culturecomprise differentiated beta-III-tubulin expressing neurons, anddifferentiated astrocytes are fewer in number or absent; or about ⅓ ofthe total number of cells in the culture comprise differentiatedbeta-Ill-tubulin expressing neurons and about ⅓ of the total number ofcells in the culture comprise differentiated oligodendrocytes, anddifferentiated astrocytes are fewer in number or absent; ordifferentiated astrocytes are present in the cell culture, and ⅓ or lessof the total number of cells in the culture comprise differentiatedneurons; or about ⅔ of the total number of cells in the culture comprisedifferentiated astrocytes, and ⅓ or less of the total number of cells inthe culture comprise differentiated neurons. In another embodiment, atleast 50%, 60%, 70%, 80% or more of total number of cells in the cultureare neurons, oligodendrocytes or astrocytes. In various aspects of theembodiments set forth herein, neurons optionally express detectableamounts of parvalbumin or GABA.

As set forth herein, isolated and purified mammalian PDGF-responsiveneural precursor (PRP) cells are distinct from neural stem cells (NSC).In one embodiment, a mammalian PDGF-responsive neural precursor (PRP)cell is more motile as compared to a progeny of EGF-responsive neuralstem cell (NSC).

Isolated and purified mammalian PDGF-responsive neural precursor (PRP)cells include primary isolates from appropriate nerve tissue or organs(e.g., brain medial ganglionic eminence). Isolated and purifiedmammalian PDGF-responsive to neural precursor (PRP) cells furtherinclude progeny cell or neurosphere of primary cell isolates.

Isolated and purified mammalian PDGF-responsive neural precursor (PRP)cells include cells that have been contacted with a factor or stimuli,or subjected to a condition, in vitro, ex vivo or in vivo. In oneembodiment, a mammalian PDGF-responsive neural precursor (PRP) cell hasbeen contacted with one or more of PDGF, BDNF, NT-3, thyroid hormone,BMP-2, CNTF, EGF or T3.

Populations of clonally expanded or self-renewed mammalianPDGF-responsive neural precursor (PRP) cells, as well asundifferentiated progeny, progenitor progeny and differentiated progeny,wherein at least a portion of the cells expresses PDGF receptor alphaare additionally provided. In one embodiment, cells or progeny cells ofthe population have been contacted with a PDGFR agonist, an FGF receptoragonist, PDGF, BDNF, NT-3, thyroid hormone, BMP-2, CNTF, EGF or T3.

Mammalian PDGF-responsive neural precursor (PRP) cell as well asundifferentiated progeny, progenitor progeny and differentiated progeny,of various species and various developmental stages are provided. Invarious embodiments, a first, second, third, fourth, fifth, sixthseventh or subsequent generation progeny cell or cells (e.g.,undifferentiated progeny, progenitor progeny and differentiated progeny)of mammalian PDGF-responsive neural precursor (PRP) cell is provided. Infurther embodiments, a cell is human, primate, murine, rattus, bovine,porcine, equine, avian, cavia, lagomorph, canine or feline, and is ofembryonic, fetal, juvenile or adult origin.

Transformed mammalian PDGF-responsive neural precursor (PRP) cells, aswell as undifferentiated progeny, progenitor progeny and differentiatedprogeny, are also provided. In one embodiment, a cell has beentransformed with a nucleic acid (encoding a protein or a homologousrecombinant construct). In particular aspects, a protein is selectedfrom a neurotransmitter, neurotransmitter receptor, growth factor (e.g.,nerve growth factor, brain-derived neurotrophic factor, neurotrophin,CNTF, amphiregulin, basic FGF, acidic FGF, EGF, transforming growthfactor-alpha, transforming growth factor-beta, PDGF, insulin-like growthfactor or interleukin), growth factor receptor (e.g., low affinity nervegrowth factor receptor, CNTF receptor, neurotrophin receptor, EGFreceptor, FGF receptor or amphiregulin receptor),neurotransmitter-synthesizing enzyme (e.g., tyrosine hydroxylase,tryptophan hydroxylase, phenylethanolamine N-methyltransferase,histidine decarboxylase, glutamic acid decarboxylase, cholineacetyltransferase, dopa decarboxylase, dopamine beta hydroxylase oramino acid decarboxylase), neurotransmitter receptor-synthesizingenzyme, growth factor-synthesizing enzyme, growth factorreceptor-synthesizing enzyme, or a neuropeptide (e.g., substance-P,neuropeptide-Y, enkephalin, vasopressin, vasoactive intestinal peptide,cholecystokinin, glucagon, bombesin, somatostatin, tachykinin, endorphinor calcitonin gene-related peptide).

In accordance with the invention, further provided are cell culturesincluding a PDGF-responsive neural precursor (PRP) cell that expressPDGF receptor alpha. In one embodiment, a cell of the culture, whencontacted with one or more of thyroid hormone, BMP-2, CNTF or T3, givesrise to a differentiated neural cell that expresses detectable amountsof one or more protein markers selected from: GABA, parvalbumin, beta-IItubulin, calbindin D, calretinin, O4, neurofilament M (NFM), myelinbasic protein (MBP), TOA-64/TUC-2 and GFAP, and the cell of the cultureis or has been contacted with one or more of PDGF, thyroid hormone,BMP-2, CNTF or T3. In another embodiment, a cell of the culture, whencontacted with one or more of thyroid hormone, BMP-2, CNTF or T3, givesrise to a differentiated neural cell that expresses detectable amountsof one or more protein markers selected from: GABA, parvalbumin, beta-IItubulin, calbindin D, calretinin, O4, neurofilament M (NFM), myelinbasic protein (MBP), TOA-64/TUC-2 and GFAP, and the cell of the cultureis or has been contacted with one or more of PDGF, thyroid hormone,BMP-2, CNTF or T3 effective to increase expression of detectable amountsof one or more protein markers selected from: GABA, parvalbumin, beta-IItubulin, calbindin D, calretinin, O4, neurofilament M (NFM), myelinbasic protein (MBP), TOA-64/TUC-2 and GFAP.

In accordance with the invention, additionally provided are cellcultures enriched for PDGF-responsive neural precursor (PRP) cells thatexpress PDGF receptor alpha. In one embodiment, at least a portion ofthe enriched cells, when contacted with one or more of thyroid hormone,BMP-2, CNTF or T3, differentiate into a neural cell that expressesdetectable amounts of one or more protein markers selected from: GABA,parvalbumin, beta-II tubulin, calbindin D, calretinin, O4, neurofilmentM (NFM), myelin basic protein (MBP), TOA-64/TUC-2 and GFAP. In anotherembodiment, at least a portion of the enriched cells, when contactedwith one or more of thyroid hormone, BMP-2, CNTF or T3, differentiateinto a neuron, oligodendrocyte, astrocyte or mixture thereof.

As used herein, the term “isolated,” when used to refer to a compositionsuch as a cell means that the composition has been removed from it'snaturally occurring environment. Such compositions need not be purifiedor homogeneous, but can be substantially free of other cell types orother cellular material with which it naturally occurs in the tissue oforigin (e.g., neural tissue). Thus, for example, an isolated primary PRPneurosphere can be substantially free of connective tissue present inbrain tissue or differentiated neural cells (e.g., neurons,oligodendrocytes, astrocytes, etc.). Accordingly, cells substantiallyfree of connective tissue and cells dissociated from other cell ortissue types are further provided, wherein the cells have or have notbeen contacted with a PDGFR agonist.

Isolated compositions can be re-introduced into its naturally occurringenvironment after removal. For example, an isolated PRP cell can beremoved, subject to clonal expansion, progenitor cell formation ordifferentiation, and be reintroduced (e.g., transplanted) into asubject.

As used herein, the term “purified,” when used to refer to a compositionsuch as a cell means that the composition has been separated fromcomponents with which it normally associated naturally occurringenvironment. A cell sample is considered “pure” when the sample has atleast 60% or more cells (e.g., 65%, 70%, 75%, 80%, 85%, 90%, 95%, ormore, 99%) than other cells of clonal origin.

As used herein, the term “enriched,” when used to refer to a compositionsuch as a cell means that the relative proportion of the composition hasbeen increased as compared to the proportion of the composition prior toenrichment. For example, a PRP cell prior to enrichment may comprise 5%of the total cell number, but will comprise greater than 5% of the totalcell number following enrichment.

Terms such as “stem cell,” “precursor cell” and “progenitor cell” arecommonly used in the art. The terminology used for undifferentiatedneural cells has evolved such that these cells are referred to generallyas “neural stem cells.” Undifferentiated neural cells do have differentcharacteristics and cell fates.

Totipotent stem cells can give rise to all cell types found in anembryo, fetus, or developed organism, including the embryonic componentsof the trophoblast and placenta required to support development andbirth. The zygote and the cells at the very early stages followingfertilization (i.e., the 2-cell stage) are considered totipotent.

Pluripotent stem cells are somewhat less plastic in theirdifferentiative capacity than totipotent stem cells, but can become allcell types that are found in an implanted embryo, fetus, or developedorganism. Unlike, totipotent stem cells, pluripotent stem cells do notform embryonic components of the trophoblast or placenta.

The term “multipotent,” when used in reference to a cell is a progeny ofa stem cell within a particular tissue, organ, or physiological system.A multipotent stem cell is able to divide for many generations (thenumber of cell divisions may or may not be limited) under certainconditions and can give rise to daughter cells (typically, at least oneis an undifferentiated cell) a proportion of which eventually terminallydifferentiates. As an example, a multipotent neural stem cell (NSC) is acell that can undergo self-renewal or clonal expansion for manygenerations, and can eventually terminally differentiate into cell typesthat are normal components of the nervous system (e.g., cells present inCNS or PNS). Differentiated neural cells include neurons,oligodendrocytes and astrocytes.

A “neural precursor cell,” as used herein, refers to an undifferentiatedcell derived from a multipotent neural stem cell (NSC), but is notitself a stem cell. One distinguishing feature of a precursor cell isthat, unlike a stem cell, it has a somewhat more limited self-renewal orclonal proliferative ability. Precursor cells can produce progeny thatare capable of differentiating into more than one cell type.

PRP cells of the invention are neural cells that can be induced toproliferate as set forth herein under conditions that allow self-renewalor clonal proliferation. PRP cells can also terminally differentiate andgive rise to different types of neural cells, oligodendrocytes, neuronsand astrocytes, under appropriate conditions or stimuli, in vitro or invivo. PRP cells can therefore be considered neural precursor cells.

A “progenitor cell,” is an early descendant of a pluri-potent ormulti-potent stem cell that can only differentiate, but typically doesnot undergo self-renewal or clonal expansion. In contrast, a stem cellor a precursor cell can renew itself (undergo cell division therebymaking more stem cell progeny) or it can differentiate (undergo celldivision and with each generation evolve into different types of cells).A progenitor cell is typically more limited into the kinds of cells itcan give rise to than a stem or precursor cell. Progenitor cells aretypically more differentiated than stem cells. Progenitor cells are alsotypically “lineage committed cell,” which is a cell that is no longerpluripotent but has been induced to differentiate into one or morespecific cell types.

Non-clonal progeny of neural stem cells and precursor cells includeprogenitor cells. The progenitor cells generated from a singlemultipotent neural stem cell are capable of differentiating intoneurons, astrocytes and oligodendrocytes. As discussed, progenitor cellshave little clonal proliferative ability and are typically committed toa particular path of differentiation and will, under appropriateconditions, eventually differentiate. An N/O cell has little if anyclonal proliferative ability, but can differentiate into differentneural cell types, namely neurons and ligodendrocytes and, therefore,can be considered a progenitor cell.

A “progeny” cell of any cells described herein refers to any and allfirst, second, third, fourth, fifth, sixth, seventh, eight, ninth,tenth, or any subsequent generation cell derived from a parental cell.Progeny of PRP cells include cells resulting from self-renewal/clonalproliferation or differentiation. Particular examples of progenytherefore include cells comprising neurospheres that form from primaryPRP cells that undergo self-renewal/clonal proliferation. Additionalparticular examples of progeny include differentiated cells derived fromneurospheres that form when PRP cells undergo cell division. Specificnon-limiting examples of such differentiated progeny include neurons,oligodendrocytes and astrocytes. Progeny of PRP cells further includeprogenitor cells, which are cells intermediate in the developmentallineage between PRP cells and a differentiated cell. A specificnon-limiting example of such a progenitor cell is an N/O cell.

As used herein, a “neurosphere” or “sphere,” refers to a cluster ofneural stem cells derived from a single parental neural cell. Neuralcells comprising the neurosphere may be self-renewed or clonallyproliferated progeny cells derived from a single parental cell. Underappropriate conditions or stimuli, neurospheres can typically bemaintained for multiple passages in vitro without appreciable formationof fully differentiated progeny cells.

A “primary neurosphere” of PRP cells is produced from brain tissue inthe presence of PDGF or other appropriate condition or stimuli. Primaryneurospheres are generated from brain tissue without cell passaging. A“secondary neurosphere” is a neurosphere generated by dissociating(passaging) a primary neurosphere and culturing dissociated cells underconditions that result in formation of neurospheres from single cells. A“tertiary neurosphere” is a neurosphere generated by dissociating(passaging) a secondary neurosphere and culturing single dissociatedcells under conditions that result in the formation of neurospheres fromsingle cells, and so forth.

Neural cells comprising a neurosphere can give rise to precursor cells,progenitor cells or differentiated progeny cells derived from a singleparental neural cell, in vitro or in vivo. For example, a differentiatedprogeny cell may comprise a cell that expresses a protein marker or hasone or more morphological characteristics of a neuron, oligodendrocyteor astrocyte. Neural cells comprising a neurosphere may give rise tointermediate progeny cells with respect to the parental multipotentneural cell and a differentiated cell arising from the intermediateprogeny. For example, an intermediate cell can be an N/O progenitorcell, which is intermediate between a PRP cell and differentiatedprogeny oligodendrocyte or neuron. In another example, an intermediatecell can be PRP cell, which is intermediate between an NSC cell anddifferentiated astrocyte. Neurospheres need not be a single cell type,but may comprise multiple precursor, intermediate (e.g., progenitor) ordifferentiated cells. For example, a neurosphere may comprise apopulation of PRP cells with or without any of N/O cells, neurons,oligodendrocytes or astrocytes.

Precursor cells, progenitor cells or differentiated progeny cells canarise in various proportions, depending upon the factors, conditions orstimuli to which the cells have been subjected to or treated, in vitroor in vivo. For example, for a differentiated cell, such as a neuron,oligodendrocyte or astrocyte, a plurality of progeny cells may compriseless or more than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, of neurons,oligodendrocytes or astrocyts, or cells that express a protein marker orhas one or more morphological characteristics of a neuron,oligodendrocyte or astrocyte.

The term “cell culture” or “culture” refers to cells grown or maintainedin an in vitro or artificial environment. A “cell culture” is a genericterm that can also be used to encompass individual clonal cells, butalso of groups of cells (e.g., neurospheres), progenitor cells,differentiated cells and mixtures thereof. A “cell culture medium,” or“culture medium” are used interchangeably to refer to a nutritivecomposition intended to maintain viability of cells.

PRP cells can be obtained from embryonic, fetal, post-natal, juvenile oradult neural tissue. The neural tissue can be obtained from any animalthat has neural tissue such as insects, fish, reptiles, birds,amphibians, mammals, etc. Typically, neural tissue suitable forobtaining PRPs is present in neural tissue of mammals, such as human andnon-human primates, dogs, cats, rodents (mice, rats, guinea pigs) andrabbits. Neural tissue can be derived from the central nervous system,for example, the brain.

Non-human animals may be euthanized, and the neural tissue and specificarea of interest removed using a sterile or non-sterile procedure. Anarea of particular interest is the ventral forebrain. The medialganglionic eminence (MGE) is one area from which PRPs are present andcan be obtained.

Human neural stem cells may be derived from embryonic or fetal tissuefollowing elective abortion, or from a post-natal, juvenile or adultdonor. Autologous neural tissue can be obtained by biopsy, or from asubject undergoing neurosurgery in which neural tissue is removed, forexample, during epilepsy surgery, temporal lobectomy orhippocampalectomy.

PRP cells obtained from donor tissue can be dissociated. Cells can bedissociated using mechanical dissociation, as set forth in Example 1, orby other methods known in the art. Such methods include, for example,treatment with enzymes such as trypsin, collagenase. Dissociation ofcells can be carried out in tissue culture medium (e.g., MHM). The cellscan be cultured on a fixed substrate or in suspension. Cells plated on afixed substrate typically have an initial density of about 1-5×10⁴cells/ ml. Cells cultured in suspension have an approximate density ofapproximately 1×10⁴ to 1×10⁵ cells/ml.

PRP cells can be dissociated from other cells or tissue. For example,PRP cells can be substantially free of other neural or non-neural celltypes present in the donor region, or free of connective tissue(connecting extracellular matrix).

Dissociated PRP cells can be maintained in culture medium capable ofsupporting cell growth, which can optionally include, supplementsrequired for cellular metabolism, such as glutamine and other aminoacids, vitamins, minerals and proteins such as transferrin and the like.Culture medium can also optionally include antibiotics to preventcontamination with bacteria, fungi (yeast, mold) or mycoplasm, such aspenicillin, streptomycin, gentamicin, fungizone, etc. Culture conditionsare at or near physiological conditions. The pH of the culture medium isclose to physiological pH, typically between pH 6-8, or between about pH7.0 to 7.8 (e.g., pH 7.4). Physiological temperatures range betweenabout 30° C. to 45° C. Cells are typically cultured at temperaturesbetween about 32° C. to about 42° C. (e.g., 37° C.).

The culture medium can be supplemented with factors, such as factorsthat modulate (increase or decrease) growth or proliferation and progenyformation. Such factors can be used to induce, promote or stimulate, orto prevent, decrease or inhibit progeny cell formation. Progeny cellformation includes clonal prolferation/self renewal, growth orproliferation or formation of intermediate (progenitor) cells, or growthor proliferation or formation of differentiated cells.

Non-limiting examples of such factors include “growth,” “survival,” or“mitogenic” factors which are molecules that alone or in combinationwith other factors can induce, promote or stimulate cell growth,survival, proliferation, differentiation, or tropism on cells or progenythereof, in vitro or in vivo. Exemplary growth factors includeplatelet-derived growth factor (PDGF-AA, PDGF-BB and PDGF-AB), acidicfibroblast growth factor (aFGF or FGF-1), basic fibroblast growth factor(bFGF or FGF-2), brain-derived neurotrophic factor (BDNF), neurotrophin3 (NT-3), EGF, SHH, amphiregulin and transforming growth factor alpha(TGFalpha).

It is understood that functionally equivalent growth and survivalfactors are also considered to be included.

Platelet derived growth factor or PDGF is a protein factor which (1)shares substantial sequence identity with the native human PDGF; and (2)possesses a biological activity of the native human PDGF. Native PDGFconsists of two polypeptide chains selected from Chain A and Chain B.Chain A and Chain B are similar. For example, the human Chain A andChain B shares 56% sequence identity in the mature PDGF molecule. A PDGFmolecule may consist of AA, AB or BB. A discussion of the structural andfunctional relationship of PDGF can be found, for example, in Hannink etal., Biochem Biophys Acta 989(1):1 (1989).

The term “substantial sequence identity,” when used in reference to aprotein, such as PDGF means there is sufficient sequence identity (e.g.,at least one polypeptide that is at least about 30% identical with ChainA or Chain B of the native human PDGF at the amino acid level, or more,40% or more at least about 60%, at least about 70%, and at least about80% identical with Chain A or Chain B of the native human PDGF at theamino acid level) that the sequence retains a biological activity ofPDGF. Thus, PDGF encompasses deletion, insertion, or substitutionmutants of native human PDGF, provided such mutants retain at least apartial activity of native human PDGF. The term PDGF thereforeencompasses PDGFs of other species, provided that the PDGF sequencesretain at least a partial activity of native human PDGF. Arepresentative “biological activity of PDGF” is binding to a PDGFreceptor and stimulating tyrosine kinase activity of the receptor (Ek etal., Nature 295(5848):419 (1982); Nishimura et al., Proc Natl Acad SciUSA 79(14):4303 (1982)).

The term “percent identity” or “% identity,” when used in reference to aprotein, such as PDGF, refers to the percentage of amino acid sequencein Chain A or Chain B of the native human PDGF which are also found inthe PDGF comparison sequence, when the two sequences are optimallyaligned (including gaps). Percent identity can be determined by methodsor algorithms known in the art, such as LALIGN or BLAST.

In addition to proliferation-inducing factors, growth factors that maybe used (in culture medium or administered in vivo) in order to modulatecell survival, growth, proliferation or differentiation of cellsinclude, for example, BMP-2, a thyroid hormone, triiodothyronine (T3),ciliary neurotrophic factor (CNTF), NGF, thyrotropin releasing hormone(TRH), transforming growth factor beta (TGFbeta) and insulin-like growthfactors (e.g., IGF₁). It is understood that functionally equivalentgrowth factors are also considered to be included.

Further non-limiting examples of growth factors and other stimuli thatcan be used to modulate cell survival, growth or proliferation andprogeny formation include FGF-1, FGF-2, neurotrophin 4 (NT-4),interleukins, leukemia inhibitory factor (LIF), cyclic adenosinemonophosphate, forskolin, tetanus toxin, high levels of potassium,glucocorticoid hormones (e.g., dexamethasone), isobutyl3-methylxanthine, somatostatin, growth hormone and retinoic acid. Theseand other functionally equivalent growth factors and stimuli areapplicable in the invention compositions and methods.

Growth factors can be used in amounts that provide the intended effect.In culture medium, typical amounts range between about 1 fg/ml to 1mg/ml. Concentrations between about 1 to 100 ng/ml are usually adequate.Titration studies can be used to determine optimal concentration of aparticular growth factor, or combination of factors.

Within about 3-4 days in a proliferation-inducing growth factor (e.g.,PDGF), a PRP cell begins to divide giving rise to a cluster ofundifferentiated clonal cells referred to as a “neurosphere.” The cellsof a single neurosphere are progeny of a single PRP cell and are clonalin nature. With continued appropriate culture conditions or stimuli,such as culturing in the presence of an appropriate growth factor (e.g.,PDGF), cells within the neurosphere continue to divide and proliferateresulting in an increase in the size of the neurosphere and the numberof clonal, undifferentiated cells therein. Under these conditions, PRPneurospheres do not appreciably differentiate and do not expressdetectable levels of one or more markers associated with differentiatedneural cells, such as gamma-aminobutyric acid (GABA), paravalbumin,beta-II tubulin, neurofilament M (NFM), O4, myelin basic protein (MBP),and glial fibrillary acidic protein (GFAP). After about 4 to 5 days,proliferating neurospheres detach from the culture dish and appear asfree-floating clusters characteristic of neurospheres.

Neurospheres can be dissociated to form single cells, counted andreplated at the desired density and passaged to reinitiateself-proliferation and clonal expansion. A percentage of thesedissociated cells form new neurospheres largely composed ofundifferentiated cells. This procedure can be repeated for subsequentgeneration of secondary neurospheres, tertiary neurospheres, and soforth until the desired number of cells, or neurospheres, are obtained.

The process by which PRP cells grow and proliferate without appreciabledifferentiation is referred to herein as “clonal-expansion,” or“self-renewal” and grammatical variations thereof. Clonal expansionrefers to cells that proliferate from a single cell that are able torenew themselves for multiple generations in vitro under appropriateconditions or stimuli. Clonal expansion and self-renewal does notrequire that the cells be capable of propagation indefinitely. Suchcells may be limited in the number of times they can be passaged beforeundergoing senescence.

Appreciable differentiation occurs when greater than 10-15% of theprogeny cells are differentiated into a particular neural cell type,such as a neuron, oligodendrocyte or astrocyte, for a given round ofcell-division, or generation. Appreciable differentiation does not referto the presence of progenitor cells, since such cells are not considereddifferentiated.

PRP cells can be proliferated in vivo or in vitro. PRP progeny cells canbe prepared by culturing appropriate brain tissue (e.g., MGE) in thepresence of PDGF, but not EGF, FGF-2, or TGF. Clonal expansion can beincreased or stimulated under appropriate conditions or stimuli. Forexample, administering a growth factor, or a combination of growthfactors to a subject, or contacting cells in vitro or in vivo with agrowth factor, or a combination of growth factors, or providingappropriate culture conditions or a stimulus. In particular, PDGF andFGF, PDGF and BDNF, and PDGF and NT-3, together, increase PRP clonalproliferation. Accordingly, cells can be proliferated in these and otherfunctionally equivalent growth factors in order to increase or stimulateclonal expansion and formation of PRP cells or neurospheres.

PRP cell differentiation can be induced as set forth herein. Forexample, BMP2- can be administered to or contacted with PRP cells invivo or in vitro in order to give rise to neurons. T3 can beadministered to or contacted with PRP cells in vivo or in vitro in orderto give rise to oligodendrocytes. BMP2 and CNTF can administered to orcontacted with PRP cells in vivo or in vitro in order to give rise toastrocytes.

Accordingly, factors can be added alone or in a combination with otherfactors, conditions or stimuli in order to produce PRP cells. Factorsand the like can also be added in a temporal sequence (e.g.,administration of, or contact with, a first growth factor, whichinfluences expression of a second growth factor receptor, followed byadministration of or contact with the second growth). For example, PRPcells can be contacted first with T3, followed by contact with. BMP-2and CNTF, which produces neurons and astrocytes.

Within about 2-3 days after PRP cells have been exposed to a factor orculture condition that can cause PRP cells to give rise todifferentiated cells, PRP differentiated progeny begin to appear.Depending on factor(s) or culture condition, progeny cells expressmarkers typically found on neurons, astrocytes or oligodendrocytes.Markers can be proteins or other molecules that are associated with orproduced by one or more neural stem cells, precursor cells, progenitorcells or differentiated cells. The pattern of markers can be used toidentify neural cell types and differentiation stage.

Exemplary cellular markers for neurons include parvalbumin,β-III-tubulin gamma-aminobutyric acid (GABA), neuron specific enolase(NSE), NF and cytoskeletal protein MAP-2. Neurotransmitters,neurotransmitter receptors and enzymes that participate inneurotransmitter synthesis, deactivation (inhibition) or uptake areoften expressed by neurons, which can be used as a marker to aid inidentifying neurons.

Specific non-limiting examples of neurotransmitters includeacetylcholine (ACh), dopamine, epinephrine, norepinephrine, histamine,serotonin or 5-hydroxytryptamine (5-HT), neuropeptides such as substanceP, adrenocorticotrophic hormone, vasopressin or anti-diuretic hormone,oxytocin, somatostatin, angiotensin II, neurotensin and bombesin,hypothalamic releasing hormones such as TRH and luteinizing releasinghormone, gastrointestinal peptides such as vasoactive intestinal peptide(VIP) and cholecystokinin (CCK) and CCK-like peptide, opioid peptidessuch as endorphins like β-endorphin and enkephalins such as met- andleu-enkephalin, prostaglandins, amino acids such as inhibitoryneurotransmitter gamma amino butyric acid (GABA), glycine, glutamate,cysteine, taurine and aspartate and dipeptides such as carnosine.

Specific non-limiting examples of neurotransmitter-synthesizing enzymesinclude glutamic acid decarboxylase (GAD) which is involved in thesynthesis of GABA, choline acetyltransferase (ChAT) for ACh synthesis,dopa decarboxylase (DDC) for dopamine, dopamine beta-hydroxylase (DBH)for norepinephrine, and amino acid decarboxylase for 5-HT. Enzymesinvolved in deactivation or inhibition of neurotransmitters includeacetyl cholinesterase (AChE), which deactivates ACh.

Enzymes involved in uptake of neurotransmitters into neuronal terminalsinclude monoamine oxidase and catechol-o-methyl transferase fordopamine, for 5-HT, and GABA transferase for GABA. Neurotransmitterreceptor markers include

AChE nicotinic and muscarinic receptors, adrenergic receptors (e.g.,alpha1, alpha2, beta1, beta2, etc.) and the dopamine receptor. Reliablemarkers useful for neuron identification include neuron specific enolase(NSE), NF, NeuN, and the neuron specific protein, tau-1.

Exemplary cellular markers for astrocytes include glial fibrillaryacidic protein (GFAP). Type 1 astrocytes, which are differentiated glialcells that have a flat, protoplasmic/fibroblast-like morphology, areimmunoreactive for GFAP but not A2B5. Type II astrocytes, which aredifferentiated glial cells that display a stellate process-bearingmorphology, are immunoreactive for GFAP as well as A2B5.

Exemplary cellular markers for oligodendrocytes include NFM, MBP, O4 andgalactocerebroside (GalC, a myelin glycolipid), a myelin glycolipididentifier. In temporal fashion, cells first become immunoreactive forO4, GalC and finally, MBP. Cells that do not express intermediatefilaments specific for neurons or for astrocytes, typically expressthese oligodendrocyte markers. These cells also possess a characteristicoligodendrocyte morphology.

The presence of such markers can be assayed by various methods known inthe art including, for example, immunocytochemistry. Antibodies to anyof the aforementioned protein markers can be used in immunocytochemistryto identify the corresponding proteins. Immunocytochemistry (e.g.dual-label immunofluorescence and immunoperoxidase methods) utilizesantibodies that detect these proteins. In situ hybridizationhistochemistry can also be performed, using nucleic acid (e.g., cDNA orRNA) probes specific for the marker mRNA. Such in situ techniques can becombined with immunocytochemical methods to enhance identification ofneural cell types. If desired, antibodies can be applied to Western andNorthern blot procedures respectively to also aid in cellidentification. Such techniques can be used to identify the cellularcharacteristics or determine phenotypic properties of neural cells suchas neurons, astrocytes and oligodendrocytes. Such techniques can also beused to determine the effect of growth factors on the differentiatingcells, as well as in screening and identification methods modulate canbe determined.

In accordance with the invention, moreover provided are in vitro and invivo methods of producing mammalian PDGF-responsive neural precursor(PRP) cells that express PDGF receptor alpha, via primary PRP cellisolation as well as progeny formation by clonal expansion orself-renewal, formation of progenitor cells, and formation ofdifferentiated cells, as well as populations of cells produced by thevarious methods. In one embodiment, a method includes culturing brainganglionic eminence (e.g., medial ganglionic eminence; MGE) in a culturemedium containing PDGF under conditions allowing clonal proliferation ordifferentiation of the PRP cells. In various aspects, a culture mediumdoes not contain EGF or FGF2; contains one or more of: PDGF, thyroidhormone, BMP-2, CNTF or T3; or contains one or more of: PDGF, BDNF, NT-3or FGF2. In additional aspects, a method includes inducing clonalproliferation or self-renewal of the PRP cells (e.g., by contacting PRPcells with PDGF and FGF-2; or PDGF and BDNF; or PDGF and NT-3). Infurther aspects, a method includes inducing formation of PRP cellneurospheres (e.g., a majority of the clonally proliferated cells arenot differentiated into neurons, oligodendrocytes or astrocytes). Inadditional aspects, a method includes inducing formation ofdifferentiated neurons, oligodendrocytes, astrocytes, or a combinationthereof.

In another embodiment, an in vivo method of increasing PRP cell numbers(e.g., in a mammal) includes administering a PDGFR agonist to an animal(e.g., in a mammal) in an effective amount for intracranial delivery ofthe PDGFR agonist (e.g., PDGF) to increase PRP cell numbers. In oneaspect, an animal (e.g., mammal) does not receive EGF or FGF. In anotheraspect, an animal (e.g., mammal) is administered FGF2, BDNF or NT-3substantially simultaneously with the PDGFR agonist to the mammal. Infurther aspects, administration is local, regional (brain) or systemic,intracranially, intravenously, intravascularly, intramuscularly,subcutaneously, intraperitoneally, topically, orally, nasally or byinhalation.

Methods of producing oligodendrocytes include, for example, in oneembodiment, culturing brain tissue from a mammal in a culture mediumwith a PDGFR agonist and allowing proliferation of PRP cells; anddifferentiating the proliferated PRP cells to produce oligodendrocytes.In one aspect, a step is performed by contacting the proliferated PRPcells with an effective amount of thyroid hormone or T3. In anotheraspect, oligodendrocytes are contacted with an effective amount of BMP-2and CNTF to produce neurons and astrocytes. In further aspects,proliferated PRP cells are clonally expanding by contacting said cellswith PDGF and FGF-2; or PDGF and BDNF; or PDGF and NT-3 prior to a step.

Methods of producing neurons include, for example, in one embodiment,culturing brain tissue from a mammal in a culture medium with a PDGFRagonist and allowing proliferation of PRP cells; and differentiating theproliferated PRP cells to produce neurons. In one aspect, a step isperformed by contacting the proliferated PRP cells with an effectiveamount of BMP-2. In other aspects, proliferated PRP cells are contactedwith PDGF and FGF-2; or PDGF and BDNF; or PDGF and NT-3 prior to a step.

Methods of producing astrocytes include, for example, in one embodiment,culturing brain tissue from a mammal in a culture medium with a PDGFRagonist and allowing proliferation of PRP cells; and differentiating theproliferated PRP cells to produce astrocytes. In one aspect, a step isperformed by contacting the proliferated PRP cells with an effectiveamount of BMP-2 and CNTF. In other aspects, proliferated PRP cells areexpanded by contacted with PDGF and FGF-2; or PDGF and BDNF; or PDGF andNT-3 prior to a step.

In vivo methods include mammals in need of increased numbers of PRPprecursor cells, progenitor progeny, or oligodendrocytes, neurons orastrocytes. Particular mammals include, for example, a mammal sufferingfrom a loss of or injury to oligodendrocytes, neurons or astrocytes; amammal afflicted with or is at risk of affliction with a neurologicaldisease or disorder, or undesirable medical condition. Non-limitingexamples of neurological diseases, disorders, and undesirable medicalconditions include neurodegenerative disease, stroke, aneurysm, brain orspinal cord injury or cranium or spinal column trauma, which can becaused by a stroke or surgery. Non-limiting examples of stroke includehemorrhagic stroke, focal ischemic stroke and global ischemic stroke.Neurological disease or undesirable medical conditions can affect eithercentral (e.g., brain or spinal cord) or peripheral nerves (e.g., one ormore of motor, sensory or autonomic nerves).

Cells of the invention, including, for example, PRP cells, N/O cells andclonally expanded or differentiated progeny thereof, may be manipulatedin order to produce modified forms. For example, PRP cells, N/O cellsand clonally expanded or differentiated progeny thereof can be“transfected” or “transformed” with a nucleic acid. Nucleic acid can beintroduced into such cells in vivo, ex vivo or in vitro. Suchgenetically modified cells into which nucleic acid has been introducedare conveniently referred to as transformed cells.

Transformed cells are useful in for expression of desirable proteins andcan be used in accordance with the invention methods, for example, totreat, ex vivo or in vivo stroke, brain or spinal cord injury or trauma,a disease or disorder, or undesirable medical condition of CNS or PNS,among other methods of the invention. For example, PRP cells may bemodified to express or to increase production of a biologically activesubstance such as a neurotransmitter or growth factor or the like.Transformed PRP cells can be clonally expanded or give rise todifferentiated cells, as set forth herein.

The term “transformed,” when used in reference to a cell (e.g., a PRPcell, or a clonally expanded or differentiated progeny thereof) does notonly refer to the particular method or technique for producing the cell,but, rather, the nature of the cell itself, i.e., a cell that has beenintentionally genetically modified. The nucleic acid may be stably ortransiently expressed by the transformed cells. Transformed cellsinclude progeny cells that are clonally expanded or have undergoneself-renewal (e.g., PRP cells that maintain their non-differentiatedstate), intermediate cells (e.g., N/O cells), or differentiated cells(e.g., neurons, oligodendrocytes or astrocytes).

Once PRP cells are obtained, neurospheres can be optionally formed,cells dissociated into single cells, plated on petri dishes in culturemedium and allowed to attach (e.g, overnight). Nucleic acid can beintroduced into PRP cells to produce transformed cells. PRP cells can bedifferentiated into neural cells, e.g., neurons, oligodendrocytes orastrocytes, as set forth herein, prior to or following introduction ofnucleic acid. Transformed PRP cells and clonally expanded/self-renewedprogeny thereof have the capacity to differentiate to produce neurons,oligodendrocytes or astrocytes, as set forth herein. Such differentiatedcell progeny are considered to also be within the meaning of atransformed cell.

Nucleic acid introduced into cells is typically part of a vector inwhich one or more expression control elements are operably linked to thenucleic acid of interest. Exemplary vectors include viral vectors, suchas an adenovirus, adeno-associated virus (AAV), retrovirus (mammarytumor virus (MMTV), lentivirus), vaccinia virus including pSCII, SimianVirus 40 (SV40), paramyxovirus (measles virus), herpes virus, RousSarcoma Virus (RSV), or papilloma virus. The term “operably linked,”when used in reference to the relationship between an expression controlelement and a nucleic acid means that the element regulatestranscription or translation of the nucleic acid sequence. Expressioncontrol elements can be operably linked to nucleic acid in cis or intrans.

Control elements that modulate expression include viral and mammalianexpression control elements. Specific non-limiting example includeretroviral long terminal repeats (LTRs), simian virus 40 (SV40),cytomegalovirus (CMV); and mammalian cell-specific promoters (e.g.,tyrosine hydroxylase).

A vector can include a nucleic acid that encodes a selectable marker.Non-limiting examples of selectable markers include puromycin, adenosinedeaminase (ADA), aminoglycoside phosphotransferase (neo, G418, APH),dihydrofolate reductase (DHFR), hygromycin-B-phosphtransferase,thymidine kinase (TK), and xanthin-guanine phosphoribosyltransferase(XGPRT). Such markers are useful for selecting stable transformants inculture.

Cells of the invention, including, for example, PRP cells, N/O cells andclonally expanded or differentiated progeny thereof, can have a targetedgene modification. Targeted gene modifications can be introduced viahomologous recombination methods known in the art. For example, ahomologous recombination vector can be prepared in which a gene ofinterest is flanked at its 5′ and 3′ ends by gene sequences that flankthe endogenous genome in the target cell. Homologous recombinationoccurs between the gene of interest carried by the vector and theendogenous gene following introduction of the vector into the targetcell. Methods for constructing homologous recombination vectors andhomologous recombinant animals from recombinant stem cells are commonlyknown in the art (see, e.g., Thomas et al., Cell 51:503 (1987); Bradley,Curr. Opin. Bio/Technol. 2:823-29 (1991); and WO 90/11354, WO 91/01140and WO 93/04169).

Methods for introducing nucleic acid into cells are known in the art.For example, a vector can be introduced using chemical, electrical ormechanical means such as liposomal or chemical mediated uptake of thenucleic acid. For example, a vector can be introduced by chemicaltransfection (DEAE dextran, calcium phosphate precipitation),electroporation, infection (e.g., recombinant viruses such asretrovirus, herpes-virus, adenovirus, adeno-associated virus,paramyxovirus), microinjection, a gene gun, cell fusion, liposomes,LIPOFECTIN™, lysosome fusion, synthetic cationic lipids, or a DNA vectortransporter. A variety of methods for producing transformed cells areknown in the art (see Maniatis et al., Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory, N.Y. 1982).

Non-limiting examples of nucleic acid types that can be introduced intocells include sequences encoding proteins such as growth factors andgrowth factor receptors; survival factors and survival factor receptors,neurotransmitters and neurotransmitter receptors, and synthesizing ordegrading (deactivating) enzymes. Specific examples of enzymes includethose participating in the synthesis or deactivation ofneurotransmitters, including amino acids, biogenic amines andneuropeptides. Additional non-limiting examples include reporter genessuch as bioluminescent proteins, e.g., green fluorescent protein andluciferase, chloramphenicol acetyltransferase, β-galactosidase andβ-lactamase.

PRP cells, N/O cells and clonally expanded, progenitor or differentiatedprogeny thereof that are genetically modified to produce a biologicalsubstance can be introduced into a subject. A biological substance canbe one that is useful for treatment of a central nervous system (CNS) orperipheral nervous system (PNS) injury or trauma, a disease or disorder,or any undesirable medical condition in which there is a deficiency ofthe substance or a risk of deficiency, or where a subject may benefitfrom the substance or the cell that produces the substance.

For example, transformed cells that secrete a growth or survival factor(a peptide, mitogen, or other molecule that induces, stimulates,increases or promotes growth, survival, proliferation ordifferentiation) or a growth or survival factor receptor can be usefulfor treatment of CNS or PNS disorders. Exemplary growth factors include,but are not limited to, PDGF, NGF, BDNF, the neurotrophins (NT-3,NT-4/NT-5), CNTF, amphiregulin, thyroid hormone, T3, FGF-1, FGF-2, EGF,TGFalpha, TGFbeta and insulin growth factors (IGFs). Exemplary growthfactor receptors include, but are not limited to, p75 low affinity NGFr,CNTFr, the trk family of neurotrophin receptors (trk, trkB, trkc), EGFr,FGFr, and amphiregulin receptors.

Cells can be genetically modified to produce neurotransmitters orneurotransmitter receptors such as serotonin, L-dopa, dopamine,norepinephrine, epinephrine, tachykinin, substance-P, endorphin,enkephalin, histamine, N-methyl D-aspartate, glycine, glutamate, GABA,ACh, etc. Cells can also be genetically modified to produceneurotransmitter-synthesizing enzymes including, for example, TH, DDC,DBH, PNMT, GAD, tryptophan hydroxylase, ChAT, and histidinedecarboxylase. Cells can additionally be genetically modified to produceneuropeptides including, for example, substance-P, neuropeptide-Y,enkephalin, vasopressin, VIP, glucagon, bombesin, CCK, somatostatin,calcitonin gene-related peptide, etc.

PRP precursor cells can be derived from transgenic animals. Such cellsderived from transgenic animals are a priori genetically modified.Various methods for producing transgenic animals are known in the art.In an exemplary method, nucleic acid (e.g., DNA) is introduced intosingle-celled fertilized eggs by direct microinjection of DNA. Othermethods include retroviral-mediated transfer, or gene transfer inembryonic stem cells. These and other techniques are described in Hoganet al., Manipulating the Mouse Embryo, A Laboratory Manual (Cold SpringHarbor Laboratory Ed., 1986).

Transformed PRP cells or clonally expanded progeny thereof can beimplanted for cell/gene therapy into the CNS or PNS of a subject in needof the biological substance produced by the genetically modified cells.Alternatively, transformed cells can be subjected to a differentiationprotocol in vitro prior to implantation. For example, transformedprecursor cells can be differentiated using any of the protocols setforth herein. Once transformed cells have differentiated, they may beassayed for expression of the desired biological substance, oroptionally directly implanted into a subject in need of the cells orbiological substance expressed by the transformed cell.

Cells of the invention including PRP cells, progeny thereof includingclonally expanded, progenitor or differentiated cells, and transformedcells, can be preserved or stored. For example, cryopreserved cells canbe stored long term until they are needed. The cells can be suspended inan isotonic solution, such as a cell culture medium, containing aparticular cryopreservant. Exemplary cryopreservants include dimethylsulfoxide (DMSO) and glycerol. Cryopreservants are typically used at aconcentration of 5-15%, usually about 8-10%, by volume. Cells are frozenand can be maintained at −10° C., −20° C. to −100° C., (e.g., about −70°C. to −80° C.).

PRP cells and clonally expanded, progenitor or differentiated progeny,which are able to clonally proliferate and expand when maintained inappropriate culture conditions, have many desirable characteristics forcells to be used in transplantation of CNS or PNS. For example, PRPcells and clonally expanded, progenitor or differentiated progeny, havenot been immortalized and are not of a tumorigenic origin. PRP cells andclonally expanded, progenitor or differentiated progeny, includingtransformed cells and progeny thereof, can therefore be used fortransplantation into the same or a different heterologous, autologous,or xenogeneic host (subject). PDGF, other growth or survival factors,conditions or stimuli can be administered prior to, simultaneously withor following cell transplantation.

It is possible to prepare PRP cells from a subject's own nerve tissue(e.g. in the case of tumor removal via surgical resection or a biopsy).Neural stem cell progeny may be generated from dissociated tissue andproliferated in vitro. Expanded precursor cells may be geneticallymodified if necessary, and transplanted into the CNS or PNS of asubject. PRP cells and clonally expanded, progenitor or differentiatedprogeny can be administered to any subject in need of such cells, and inany manner.

PRP cells and clonally expanded, progenitor or differentiated progenycan be used to repair damage of tissues and organs resulting frominjury, trauma, a disease or disorder, age, or any undesirable medicalcondition in which a subject may obtain a benefit. A subject can beadministered a population of PRP cells or progeny thereof to regenerateor restore neural tissues or organs which have been damaged as aconsequence of injury, trauma, a disease or disorder, age, or anyundesirable medical condition in which a subject may obtain a benefit. Asubject at risk of an injury, trauma, a disease or disorder, age, or anyundesirable medical condition in which a subject may obtain a benefitcan be administered a population of PRP cells or progeny thereof toprevent or inhibit injury, trauma, damage of neural tissues or organswhich may be a consequence of injury, trauma, damage, a disease ordisorder, age, or any other appropriate condition in which a subject mayobtain a benefit. PRP cells and progeny thereof can therefore be used inneural tissue regeneration or a replacement therapy or protocol, ex vivoor in vivo.

PRP cells and progeny thereof can be used to provide biologicalsubstances to a subject in need thereof, i.e., a subject having adeficiency of the biological substance (e.g., a growth or survivalfactor, an enzyme, neurotransmitter, etc.), a subject at risk of havinga deficiency of the biological substance, or a subject in whichproviding the biological substance will in turn provide the subject withsome objective or subjective benefit. Suitable PRP cells and progenythereof for invention methods therefore further include transformedcells, which can be used as a carrier to introduce a gene into a subjectwhich will in turn provide the subject with some objective or subjectivebenefit.

In accordance with the invention, yet additionally provided, are methodsof treating or ameliorating a disease, disorder or undesirable medicalcondition associated with insufficient numbers of or PRP loss,insufficient numbers of or neural progenitor cell loss, or insufficientnumbers of or neuron, oligodendrocytes or astrocyte loss, injury ordysfunction. Methods of the invention include reducing progression,severity, frequency, duration, susceptibility or probability of thedisease, disorder or undesirable medical condition associated withinsufficient numbers of or PRP loss, insufficient numbers of or neuralprogenitor cell loss, or insufficient numbers of or neuron,oligodendrocytes or astrocyte loss, injury or dysfunction. In oneembodiment, a method includes transplanting an effective amount of PRPcells, or any progeny thereof, to a mammal harboring the disease,disorder or medical condition. In various aspects, a method includesadministering to a mammal one or more agents selected from PDGF; PDGFand FGF-2; PDGF and BDNF; PDGF and NT-3; thyroid hormone; T3; BMP-2;BMP-2 and CNTF. In another embodiment, a method includes administeringan effective amount of PDGFR agonist to a mammal harboring the disease,disorder or medical condition, as well as one or more of FGF-2, thyroidhormone, T3, BMP-2 or CNTF.

Undesirable medical conditions include, for example, a neurologicalinjury or trauma, that affects CNS (e.g., brain or spinal cord) or PNS(e.g., one or more of motor, sensory or autonomic nerves). Non-limitingexamples of neurological injury or trauma include stroke, aneurysm,brain or spinal cord injury and cranium or spinal column trauma orinjury. Non-limiting examples of types of stroke include hemorrhagicstroke, focal ischemic stroke or global ischemic stroke.

Specific non-limiting examples of diseases, disorders and undesirablemedical conditions treatable in accordance with the invention includeAlzheimer's Disease, multiple sclerosis (MS), macular degeneration,glaucoma, diabetic retinopathy, peripheral neuropathy, Huntington'sDisease, amyotrophic lateral sclerosis (ALS), Parkinson's Disease,depression, epilepsy, neurosis and psychosis.

A “neural disease or condition associated with neuron or oligodendrocyteloss or dysfunction” is a disease or medical condition that is caused byor otherwise associated with neuron/oligodendrocyte loss or dysfunction.Examples of these diseases or conditions include neurological andneurodegenerative disorders and diseases, brain injuries or CNS or PNSdysfunctions. Neurodegenerative diseases include, for example,Alzheimer's Disease, multiple sclerosis (MS), macular degeneration,glaucoma, diabetic retinopathy, peripheral neuropathy, Huntington'sDisease, amyotrophic lateral sclerosis, and Parkinson's Disease. Braininjuries include, for example, stroke (e.g., hemorrhagic stroke, focalischemic stroke or global ischemic stroke) and traumatic brain injuries(e.g. injuries caused by a brain surgery or physical accidents). CNSdysfunctions include, for example, depression, epilepsy, neurosis andpsychosis.

In the methods of the invention in which cells are delivered in vivointo a subject, a growth or survival factor (e.g., PDGF, BMOP-2, CNTF,thyroid hormone, T3, EGF, FGF, SHH, Bc1-2, etc.), condition or otherstimuli can also be administered prior to, concurrently with, orfollowing in vivo cell delivery. A microfabricated device or implant canalso be used to deliver a growth or survival factor (e.g., PDGF, BMOP-2,CNTF, thyroid hormone, T3, EGF, FGF, SHH, etc.), condition or otherstimuli prior to, concurrently with, or following in vivo cell delivery.

PRP cells and progeny thereof are also suitable for treatingdemyelination diseases. Undifferentiated PRP cells can be clonallyexpanded as set forth herein and injected into a demyelinated targetregion. The transplanted cells are expected to differentiate in vivo.Oligodendrocytes derived from PRP cells following proliferation ordifferentiation in vitro may be injected into demyelinated targetregions in the subject.

Non-limiting examples of demyelination diseases include, for example,multiple sclerosis (MS), perivenous encephalomyelitis, neuromyelitisoptica, concentric sclerosis, acute, disseminated encephalomyelitides,post encephalomyelitis, postvaccinal encephalomyelitis, acutehemorrhagic leukoencephalopathy, progressive multifocalleukoencephalopathy, idiopathic polyneuritis, diphtheric neuropathy,Pelizaeus-Merzbacher disease, neuromyelitis optica, diffuse cerebralsclerosis, central pontine myelinosis, spongiform leukodystrophy, andleukodystrophy (Alexander type).

Cells delivered in vivo, for example, via transplantation, can bedelivered locally, regionally or systemically. Transplantation can bedone in a manner in which particular neural tissues or organs, orregions of neural tissues or organs, are targeted. For example, specificbrain regions which are affected by trauma, injury or stroke,neurodegenerative diseases, disorders or medical conditions, as setforth herein (e.g., Alzheimer's Parkinson's, aging, etc.) can betargeted for cell transplantation. Exemplary target area of braininclude the subventricular zone, which is significantly reduced in agedmice. In addition, the subventricular zone is the source of olfactoryneurons, and olfactory dysfunction is a hallmark of forebrainneurodegenerative diseases, such as Alzheimer's, Parkinson's andHuntington's diseases. An additional exemplary target area of brainincludes basal ganglia (e.g., caudate and putamen), the nucleus basalisand the substantia nigra.

Cells are administered by any appropriate technique, such as injection,via a cannula, for example. Injection methods are known in the art (see,e.g., Duncan et al., J Neurocytology, 17:351 (1988); and in NeuralGrafting in the Mammalian CNS, (Bjorklund and Stenevi, Eds. 1985)).Standard stereotactic neurosurgical methods can be used to inject cellsuspensions into the brain or spinal cord.

Cells delivered in vivo in a subject can be examined for survival usingvarious non-invasive scans such as computerized axial tomography (CATscan or CT scan), nuclear magnetic resonance (NMR), magnetic resonanceimaging (MRI) or positron emission tomography (PET). Examination ofgraft survival can be done by removing a section of neural tissue, andvisually examining the affected region.

Cells delivered in vivo in a subject can also be identified by priorincorporation of detectable markers in the cells prior totransplantation. For example, tracer dyes such as rhodamine- orfluorescein-labelled microspheres, fast blue, bisbenzamide orhistochemical markers such as the lac Z gene which produces betagalactosidase can be used to obsery the cells and ascertain theirsurvival, proliferation, differentiation, and so forth.

Activity or function of cells delivered in vivo can be assessed by usingappropriate clinical indicia. For example, various functions includingbut not limited to endocrine, motor, cognitive and sensory functions canbe ascertained in order to determine whether the cells delivered in vivohave activity or function in the subject. Motor tests include measuringmovement, balance, coordination, akinesia or lack of movement, rigidityand tremors. Cognitive tests include various tests of ability to performeveryday tasks, as well as various memory tests.

An “effective amount” is an amount sufficient to achieve the intendedpurpose. In the methods of the invention in which a detectable result orbeneficial effect is a desired outcome, such as a therapeutic benefit ina subject treated in accordance with the invention, cells can beadministered in sufficient or effective amounts. An “amount sufficient”or “amount effective” includes an amount that elicits any desirableoutcome for any duration of time and for any subjective or objectivedegree.

As used herein, an “amount sufficient” or “amount effective” refers toan amount of a PRP cells or progeny alone, or in combination with one ormore other agents or therapeutic or treatment protocols or regimens setforth herein or appropriate for the disease, provides a long or shortterm detectable response, a desired outcome or beneficial effect in agiven subject of any measurable or detectable degree or duration (e.g.,for minutes, hours, days, months, years, or cured).

An amount sufficient or an amount effective can but need not be providedin a single administration and can but need not be administered alone(i.e., without a second drug, agent, treatment or therapeutic regimen orprotocol), or in combination with another compound, agent, treatment ortherapeutic regimen. In addition, an amount sufficient or an amounteffective need not be sufficient or effective if given in single ormultiple doses without a second compound, agent, treatment ortherapeutic regimen, since additional doses, amounts or duration aboveand beyond such doses, or to additional drugs, agents, treatment ortherapeutic regimens may be included in order to be effective orsufficient in a given subject. An amount sufficient or an amounteffective need not be effective in each and every subject, nor amajority of subjects in a given group or population. Thus, an amountsufficient or an amount effective means sufficiency or effectiveness ina particular subject, not a group or the general population. As istypical for such methods, some subjects will exhibit a greater or lessresponse to a method of the invention, including treatment/therapy.

Reducing, inhibiting decreasing, eliminating, delaying, halting orpreventing a progression or worsening or an adverse symptom of thecondition, disorder or disease is a satisfactory outcome. The doseamount, frequency or duration may be proportionally increased orreduced, as indicated by the status of the condition, disorder ordisease being treated, or any adverse side effects of the treatment ortherapy. Dose amounts, frequencies or duration also consideredsufficient and effective are those that result in a reduction of the useof another drug, agent, treatment or therapeutic regimen or protocol.

An “amount sufficient” or “amount effective” includes reducing,preventing, delaying or inhibiting onset, reducing, inhibiting,delaying, preventing or halting the progression or worsening of,reducing, relieving, alleviating the severity, frequency, duration,susceptibility or probability of one or more adverse or undesirablesymptoms associated with the condition, disorder or disease of thesubject. In addition, hastening a subject's recovery from one or moreadverse or undesirable symptoms associated with the condition, disorderor disease is considered to be an amount sufficient or effective.Various beneficial effects and indicia of therapeutic benefit are as setforth herein and would be known to the skilled artisan.

An “amount sufficient” or “amount effective,” in the appropriatecontext, can refer to therapeutic or prophylactic amounts.Therapeutically or prophylactically sufficient or effective amounts meanan amount that detectably improves the condition, disorder or disease,by one or more objective or subjective clinical endpoints appropriatefor the condition, disorder or disease.

Methods of the invention therefore include providing a detectable ormeasurable beneficial effect or therapeutic benefit to a subject, or anyobjective or subjective transient or temporary, or longer-termimprovement (e.g., cure) in the condition, disorder or disease. Thus, asatisfactory clinical endpoint is achieved when there is an incrementalimprovement in the subjects condition or a partial or complete reductionin the severity, frequency, duration or progression of one or moreassociated adverse symptoms or complications or inhibition, reduction,elimination, prevention or reversal of one or more of the physiological,biochemical or cellular manifestations or characteristics of thecondition, disorder or disease.

A therapeutic benefit or improvement (“ameliorate” is used synonymously)therefore need not be complete ablation of any or all adverse symptomsor complications associated with the condition, disorder or disease butis any measurable or detectable objectively or subjectively meaningfulimprovement in the condition, disorder or disease. For example,inhibiting a worsening or progression of the condition, disorder ordisease, or an associated symptom (e.g., slowing or stabilizing one ormore symptoms, complications or physiological or psychological effectsor responses), even if only for a few days, weeks or months, even ifcomplete ablation of the condition, disorder or disease, or anassociated adverse symptom is not achieved is considered to bebeneficial effect.

As used herein, the term “subject” includes animals, typically mammaliananimals, such as but not limited to humans, non-human primates (apes,gibbons, chimpanzees, orangutans, macaques), domestic animals (dogs andcats), farm animals (horses, cows, goats, sheep, pigs), birds andexperimental animals (mouse, rat; rabbit, guinea pig).

Subjects include animal disease models (e.g., stroke, neurologicalinjury or trauma, neurodegenerative diseases, disorders or undesirablemedical conditions). Subjects include naturally occurring ornon-naturally occurring mutated or non-human genetically engineered(e.g., transgenic or knockout) animals. Subjects further include animalshaving or at risk of having a disorder or disease as set forth herein.

Subjects having or at risk of having a disorder or disease or acondition appropriate for treatment as set forth herein include subjectswith an existing condition or a known or a suspected predispositiontowards developing a symptom associated with the condition, disorder ordisease. Thus, the subject can have an active acute or chroniccondition, disorder or disease, or a latent condition, disorder ordisease. At risk subjects include those at risk or predisposed towardssuffering from such conditions, disorders or diseases based upon geneticpredisposition or a family history, detection of increased risk, orexhibit relevant correlating symptoms indicating predisposition orsusceptibility, but the condition, disorder or disease may not presentlymanifest itself in the subject. Particular non-limiting examples ofsubjects include subjects having or at risk of having Alzheimer'sDisease, multiple sclerosis (MS), macular degeneration, glaucoma,diabetic retinopathy, peripheral neuropathy, Huntington's Disease,amyotrophic lateral sclerosis and Parkinson's Disease, depression,epilepsy, neurosis and psychosis.

As used herein, the term “associated with,” when used in reference tothe relationship between a symptom and a condition, disorder or disease,means that the symptom is caused by the condition, disorder or disease,or is a secondary effect of the condition, disorder or disease. Asymptom that is present in a subject may therefore be the direct resultof or caused by the condition, or may be a secondary effect, forexample, a subject reacting or responding to the condition, disorder ordisease.

PRP cells and progeny thereof can be included in pharmaceuticallyacceptable carriers and excipients, i.e., pharmaceutical compositions.Pharmaceutical compositions can be delivered via any route, such asintracranially, intravenously, parenterally, intrathecally,intravascularly, intramuscularly, transdermally, intradermally,subcutaneously, intranasally, or intraperitoneally. Pharmaceuticalcompositions can be delivered into the central nervous system locally orregionally, for example, by injection or infusion. Alternatively,Pharmaceutical compositions can be delivered into the central nervoussystem systemically.

Pharmaceutical compositions can include a compund that facilitatestraversal of the blood brain barrier. Blood brain barrier permeabilizersinclude, for example, bradykinin and bradykinin agonists (e.g., U.S.Pat. Nos. 5,686,416; 5,506,206 and 5,268,164).

Pharmaceutical compositions can be prepared by mixing cells with anappropriate vehicle suitable for the intended route of administration.Particular non-limiting examples of suitable carriers and excipientsinclude artificial cerebral spinal fluid, and liquids compatible withmaintaining cell viability. Liquid forms in which cells may beincorporated for administration include aqueous solutions.

Methods for screening and identifying modulators (agents, conditions orstimuli that modulate) of neural cell self-renewal/clonal expansion,progenitor progeny formation and differentiation are yet additionallyprovided. In one embodiment, a method of identifying an agent thatmodulates clonal proliferation or self renewal or differentiation of aneural precursor cell includes: providing PRP cells of or progeny cellsthereof; contacting the cells with a candidate agent; and determining ifthe candidate agent modulates clonal expansion or differentiation of thecells. In various aspects, formation of neuorspheres is determined. Inadditional aspects, differentiation into one or more of neurons,oligodendrocytes or astrocytes is determined.

Such screening and identification systems allow any agent, condition orstimuli to be screened for their ability to affect PRP or N/O cellself-renewal/clonal expansion or differentiation. Such an assay wouldinclude exposing PRP or N/O cells, as single cells, neurospheres, ormixtures (with or without progeny cells, intermediate cell ordifferentiated cells) to a particular agent (e.g., potentially bioactivesubstance), culture condition (varying cell density, substrate materialor coating, feeder layers, growth medium type, conditioned ornon-conditioned media, etc.), environmental stimuli (e.g., pH,temperature, hyper- or hypoxia), then determining whether that exposuremodulated PRP or N/O cell self-renewal/clonal expansion ordifferentiation. Detection of a change in the rate, frequency or amountof self-renewal/clonal expansion or differentiation in the presence ofthe agent, culture condition, or environmental stimulus would identifythat particular agent, culture condition, or environmental stimulus as amodulator of PRP or N/O cell self-renewal/clonal expansion ordifferentiation. For example, a change in PRP proliferation can bedetected by an increase or decrease in the number of neurospheres thatform or the size of the neurospheres.

Such methods for screening and identifying are not limited topluripotent or precursor cells. In this regard, differentiated cells,including, for example, neurons, oligodendrocytes, and astrocytes maysimilarly be employed in the assay methods to identify a modulator ofself-renewal/clonal expansion or differentiation.

Methods for screening and identifying modulators may be performed insolid phase, in solution, in culture (a primary cell isolate or cells inan in vitro culture medium, or any progeny cells thereof). Screeningmethods may be performed in vivo in appropriate animals, such as mice.

Any substance is appropriate for screening and identifying modulators.Particular non-limiting examples polypeptides and peptidomimetics,naturally occurring or recombinant, nucleic acids such as DNA or RNA.Non-protein molecules may be naturally occurring or chemicallysynthesized and include, for example, small organic compounds.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention relates. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described herein.

All publications, patents and other references cited herein areincorporated by reference in their entirety. In case of conflict, thepresent specification, including definitions, will control.

As used herein, the singular forms “a”, “and,” and “the” include pluralreferents unless the context clearly indicates otherwise. Thus, forexample, reference to “a PRP cell or progeny cell” includes a pluralityof PRP cells or progeny cells; and reference to “a symptom” includes aplurality of symptoms (e.g., adverse or undesirable). Of course, thisdoes not preclude limiting certain embodiments of the invention tospecific PRP cells or progeny cells, particular symptoms, particularconditions, disorders or diseases, particular subjects, etc., usingappropriate language.

The invention is generally disclosed herein using affirmative languageto describe the numerous embodiments. The invention also specificallyincludes embodiments in which particular subject matter is excluded, infull or in part, such as substances or materials, method steps andconditions, protocols, procedures, assays or analysis disclosed herein.Thus, even though the invention is generally not expressed herein interms of what the invention does not include, aspects that are notexpressly included in the invention are nevertheless expressly orinherently disclosed herein. Furthermore, the invention includesembodiments which exclude subject matter that, in view of the subjectmatter and relevant technology, would be incompatible with one or moreembodiments of the invention.

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, the following examples are intended to illustrate but notlimit the scope of invention described in the claims.

EXAMPLES Example 1

This example provides a description of materials and methods.

Animals. TgN(GFPU)5Nagy (GFP) mice were obtained from Jackson Laboratory(Bar Harbor, Me.) and along with CD-1 mice stocks were maintained in theUniversity of Calgary Bioscience Animal Resources Center.

Cell culture. The culture medium (MHM) was composed of DMEM/F-12 (1:1)including HEPES buffer (5 mM), glucose (0.6%), sodium bicarbonate (3mM), glutamine (2 mM), insulin (25 μg/ml), transferrin (100 μg/ml),progesterone (20 nM), putrescine (10 μM), and sodium selenite (30 nM;all from Sigma, St. Louis, Mo., except glutamine from Invitrogen,Carlsbad, Calif.). The lateral, medial, or both ganglionic eminences(LGE, MGE or both) were removed from Embryonic Day 14 (E14) mouseembryos and mechanically dissociated with a fire-polished Pasteurpipette in MHM. Cells were plated at a density of 0.01×10⁶cells/mlunless otherwise indicated.

For neurosphere generation, PDGF-AA (100 ng/ml; Peprotech, Rocky Hill,N.J.), PDGF-BB (100 ng/ml; Peprotech), EGF (20 ng/ml; Peprotech), FGF2(20 ng/ml; R&D [Minneapolis, Minn.])+heparan sulfate (2 μg/ml; HS; R&D),SHH (2 μg/ml; R&D), cyclopamine (5 μM; Toronto Research Chemicals, NorthYork, Ontario), and/or DMSO (0.1%; carrier) was added to the MHM. MHMused to generate neurospheres also contained 2% B27 (Invitrogen).

Neurospheres were differentiated on poly-L-ornithine coated coverslipsin MHM and in the presence or absence of 1% FBS (Invitrogen), BMP-2(Genetics Institute; Cambridge, Mass.), T3 (Sigma), and/or CNTF(generated as previously described (Gupta et al., J Neurobiol 23:481(1992)) for 2 to 3 DIV. In order to determine whether PDGF-inducedneurosphere generation was the result of clonal expansion, dissociatedE14 MGEs from GFP and CD1 albino mice and were cultured in PDGF-AA, 1:1,at a density of 0.02×10⁶ cells/ml for 6 DIV. The number of GFP, non-GFP,and chimeric GFP expressing neurospheres were counted after 6 DIV usinga Leica Microsystems DMIL inverted fluorescence microscope (RichmondHill, ON).

Self-renewal capacity was examined by single sphere dissociation.Briefly, single 6 DIV neurospheres of equivalent size that weregenerated in the presence of EGF, PDGF-AA, or PDGF-AA and SHH weretransferred into 96 well plates and mechanically dissociated.Dissociates were cultured in MHM supplemented with EGF, FGF2 (includes 2μg/ml HS), FGF2 and SHH, PDGF-AA, PDGF-AA and SHH, PDGF-AA and FGF2,PDGF-AA and FGF2 and SHH, PDGF-AA and DMSO (0.1%), PDGF-AA and SHH, orPDGF-AA and FGF2 and cyclopamine. The number of secondary neurospheresgenerated was counted after 9 DIV.

To determine if extrinsic factors could promote self-renewal of PRPs,individual, 7 DIV, GFP-expressing, PDGF-generated neurospheres wereisolated, dissociated in the presence of PDGF-AA, and differentiated oncoverslips that had been plated 2 days earlier with or without 0.2×10⁶cells/ml of EGF-generated cells from dissociated primary EGFneurospheres. EGF-generated feeder cells had been allowed todifferentiate for 2 days in the presence of 1% FBS. Plates were rinsed3× with MHM prior to the addition of GFP-expressing, PDGF-generateddissociates. Numbers of adherent clones and cells per clone wereassessed by GFP expression. All images were captured with a PhotometricsCoolsnap digital camera (Tuscon, Ariz.) mounted on a Leica MicrosystemsDMIL inverted fluorescence microscope with Coolsnap V1.2.0 software.

Immunofluorescence. Six DIV primary PDGF-AA-generated neurospheres weredifferentiated on poly-L-ornithine coated coverslips and after 2 DIVwere fixed for 20 min. in 4% paraformaldehyde. For mouse IgM anti-O4(1:10; Chemicon; Temecula, Calif.), coverslips were incubated in PBS (pH7.5) overnight at 4° C. Coverslips were also incubated with mouseanti-β-III-tubulin (Sigma; 1:1000), rabbit anti-GFAP (BTI, Stoughton,Mass.; 1:300), mouse anti-GFAP (Chemicon 1:500), goat anti-mouse PDGFRα(1:10; R&D), rabbit anti-GFP (1:100; Santa Cruz; Santa Cruz, Calif.),rabbit anti-Human MBP (1:200; DAKO; Mississauga, ON), mouseanti-neurofilament M (1:50; RMO270; gift from Dr. Virginia Lee), and/orrabbit anti-OLIG2 (1:250) in 0.3% Triton-X-100 in PBS for 2 hours at 37°C. After incubation with primary antibodies, all tissue was incubatedfor 1 hour in PBS and 10% normal serum of the secondary antibody host(all secondary antibodies and reagents from Jackson Immunoresearch,except for HRP-conjugated secondary from Chem icon). This was followedby a 1-hour incubation with a biotin-conjugated secondary antibody andafterwards a 1-hour incubation at 37° C. with streptavadin-Cy3 (1:1000)or streptavadin-FITC (1:500) for O4 staining. For the other primaryantibodies, the coverslips were incubated with the appropriate secondaryantibody and/or Hoechst 33258 (1:100-1000; Sigma).

The neuronal phenotypes of PDGF-AA generated progeny were examined in 6DIV neurospheres differentiated in 1% FBS for 2 DIV or on E14dissociated whole brains plated on poly-L-ornithine coated coverslipsfor 5 DIV in the presence of 1% FBS. Coverslips were incubated overnightat 4° C. in rabbit anti-rat parvalbumin (1:1000; Swant; Bellinzona,Switzerland), rabbit anti-mouse GABA (1:500; Sigma), rabbit anti-mousecalretinin (1:1000; Swant), mouse anti-mouse calbindin-D (1:200; Sigma)and/or mouse anti-β-III-tubulin. Coverslips were then incubated withappropriate secondary antibodies as above.

For immunohistochemistry on cryosections, E14 brains were dissected outand processed as previously described (Shimazaki et al., J Neurosci21:7642 (2001)). For staining with rabbit anti-mouse PDGFRα (SantaCruz), transverse sections (10 μm) were first incubated in 1% H₂O₂ inPBS for 30 minutes at RT. Subsequently, sections were incubated with theantibody (1:300) in 0.3% Triton X-100, 10% normal goat serum in PBSovernight at RT. Sections were then washed and incubated with theappropriate horseradish peroxidase-conjugated secondary antibody for 1hour at RT. Sections were then incubated with 3,3′-diaminobenzidine(Sigma; 1×10 mg tablet in 20 ml of PBS and 10 μl of 30% H₂O₂) 10 minutesor until the desired intensity of reaction product was reached. Fordouble labeling, E14 brains were fixed as above, and 10-15 μm transversesections were cut on a vibratome (Leica), mounted onto slides, and wereincubated with sheep anti-mouse EGFR (1:50; Biodesign

International, Kennebunk, Me.), or rabbit anti-mouse FGFR2 (1:50; SantaCruz) in PBS. This was followed by washes and incubation with theappropriate biotin-conjugated secondary antibodies for 2 hours at RT.Sections were then washed and incubated with streptavadin-cy3 (1:1000),followed by a 2-hour incubation at 37° C. with goat anti-mouse PDGFRα(1:10; R&D) in 0.3% triton X-100 in PBS. After washes in PBS, sectionswere incubated for 1 hour with the appropriate secondary antibody. Allimmunofluorescent slides were mounted with Fluorsave (Calbiochem; SanDiego, Calif.). Images were captured with a Photometrics Quantix cameraor an Axiocam (Zeiss; Thornwood, N.Y.) mounted on a Zeiss Axioplan2.

Example 2

This example includes data indicating that E14 medial ganglioniceminence (MGE) is the source of neurosphere generating PRPs. Thisexample also includes data indicating that PRPs have potential todifferentiate into neurons and oligodendrocytes

PDGFRα is one of the earliest markers of OLPs, and signaling by PDGF-AAis required for the generation of most oligodendrocytes (Fruttiger etal., Development 126:457 (1999)). Increasing concentrations of PDGF-AAwere used to determine whether stimulation of dissociated E14 medial andlateral ganglionic eminences (MGE and LGE, respectively) results ingeneration of neurospheres. The neurosphere assay was used becausemanipulation of primary cells is minimal compared to the immunopanningprocedures used to isolate O-2A progenitors.

PDGF-AA induced neurosphere production in a dose-dependent manner.Significantly more neurospheres were produced in 100 ng/ml of PDGF-AAcompared to all other concentrations tested (p<0.01; 12±1 neurospheresper 10,000 plated cells; Tukey HSD; n=3) (FIG. 1A).

Expression of PDGFRα is largely restricted to the MGE at E14(Tekki-Kessaris et al., Development 128-2545 (2001)). If endogenous PRPswere being isolated then their generation should be restricted to theMGE. The-studies indicate that the MGE produced significantly moreneurospheres (>4-fold; FIG. 1B) than the LGE with either PDGF-AA orPDGF-BB (p<0.0001; t test; n=4 and n=3, respectively), corroboratingthat endogenously generated PRPs were in fact isolated from MGE.

PDGF is also known to have chemotaxic effects on cortical NSCs(Forsberg-Nilsson et al., J Neurosci Res 53:521 (1998)), and thus it waspossible that neurospheres generated under PDGF stimulation resultedfrom the directed migration of NSCs along the culture dish into clumpsthat resembled clonally-derived neurospheres. This possibility wasstudied, as previously described (Represa et al., Eur J Neurosci 14:452(2001)), by culturing dissociated E14 MGEs from CD1 and TgN(GFPU)5Nagy(ubiquitous green fluorescent protein [GFP]-expressing) mice together,1:1, at 20,000 cells/ml, and in the presence of 100 ng/ml of PDGF-AA. Ifcell clumping generates the majority of the neurospheres, then mostneurospheres should contain both GFP- and non-GFP-expressing cells.However, 95±11% of the neurospheres were not chimeric for GFP expression(p<0.01; Tukey HSD; n=3) (FIG. 1C-D), and there was no difference in thenumber of GFP- or non-GFP-expressing neurospheres produced (p>0.65;Tukey HSD; n=3) (FIG. 1D). Together, the data indicate that neurospheresgenerated by PDGF stimulation are products of clonal cell proliferation.

The phenotype potential of the PDGF-generated neurospheres was examinedwith indirect immunocytochemistry using antibodies directed against GFAP(astrocytes), β-III-tubulin (neurons), and O4 (oligodendrocytes).PDGF-generated neurospheres differentiated into neurons and/oroligodendrocytes, but not astrocytes, after 2 DIV in the presence of 1%FBS (FIG. 1E).

The MGE largely gives rise to interneurons that migrate out towards thecortex, in a manner similar to OLPs (Marin et al., Nat Rev Neurosci2:780 (2001)). To determine if PDGF-generated neurons, which areMGE-derived, expressed interneuronal antigens, immunocytochemistry withantibodies directed against GABA, calbindin D, calretinin andparvalbumin, was used to examine the, phenotype of the differentiatedneurons (after 2 DIV in the presence of 1% FBS) from 6 DIVPDGF-AA-generated neurospheres. The studies indicate that all antigenswere detected in E14 dissociated whole brains differentiated for 5 DIV.However, differentiated neurons from PDGF-AA-generated neurospheres,identified by immunoreactivity, expressed only GABA or parvalbumin (FIG.1F). These findings are in agreement with transplantation studies byWichterle et al., Development 128:3759 (2001), which demonstrated thatover 70% of the neurons derived from the MGE differentiated intoparvalbumin-immunoreactive, GABAergic interneurons.

To determine if neurons clearly differentiated from the progeny of PRPsin vivo, co-expression of neuronal antigens in PRPs in vivo wasascertained. PDGFRα-immunoreactive cells within the E14 MGE were alsoimmunopositive for TOAD-64/TUC-2 (Minturn et al., J Comp Neurol 355:369(1995)), an early neuron-specific antigen (FIG. 1G). Together, the dataindicate that PRPs contribute, in addition to oligodendrocytes, to thegeneration of neurons within the forebrain.

Example 3

This example includes data indicating that PRPs are distinct fromEGF-responsive NSCs.

The finding that PRPs reside mainly in the MGE, and that they do notproduce astrocytes, indicate that PRPs are distinct from EGF-responsiveNSCs. Indeed, EGF can generate neurospheres from both MGE and LGE, andthese neurospheres produce neurons, oligodendrocytes, and astrocyteswhen differentiated in 1% FBS (Reynolds et al., Dev Biol 175:1 (1996)).If PDGF and EGF stimulate distinct populations to produce neurospheres,one would predict a predominantly non-overlapping pattern of PDGF andEGF receptor expression within the MGE or anterior entopeduncular (AEP).Thus, the expression of PDGFRα and EGFR was studied in transversesections of the E14 forebrain.

PDGFRα expression was largely restricted to the AEP, preoptic area, theprimordia of the choroid plexus, and the meninges (FIG. 2A).Double-labelling for PDGFRα (arrowhead) and EGFR (arrow) revealed twopopulations of cells that were non-overlapping in their expression ofthese receptors (FIG. 2B-D).

Tropepe et al., Dev Biol 208:166 (1999), found that embryonic EGF andFGF NSCs were two distinct cell populations, by virtue of theirgeneration of neurospheres being additive under clonal conditions. Thus,if PRPs and EGF-responsive NSCs are truly different populations asindicated by the expression patterns of their receptors, the generationof neurospheres with both PDGF-AA and EGF should also be additive.Accordingly, cells from the LGE or MGE were cultured (10,000 cells/ml)in the presence of 100 ng/ml of PDGF-AA, 20 ng/ml of EGF, or both, andthe resultant primary neurospheres were counted. Dissociated MGEscultured in the presence of EGF and PDGF-AA produced significantly moreneurospheres than MGEs cultured in either EGF or PDGF-AA alone (FIG. 2E;p<0.05; LSD test; n=4). In contrast, there was no difference in thenumber of neurospheres produced from dissociated LGEs cultured in thepresence of EGF and PDGF-AA in comparison to EGF alone (p>0.86; LSDtest; n=4).

Primary EGF-generated neurospheres, when dissociated and cultured inEGF, always produce many secondary neurospheres (Reynolds et al., DevBiol 175:1 (1996)), indicative of their extensive self-renewal capacity.PRPs were therefore studied for a similarly extensive capacity forself-renewal. Single, primary PDGF-generated neurospheres producedalmost no secondary neurospheres (1±1) when mechanically dissociated in96-well plates containing PDGF-AA (FIG. 2F; n=7, 69 neurospheresexamined [NE]). Primary PDGF-generated neurospheres passaged into EGF(n=3, 32 NE), also produced very few (3±1) secondary neurospheres. Incontrast, primary EGF-generated neurospheres processed in the samemanner, but passaged into PDGF-AA (*p<0.0001; Tukey HSD; n=3, 24 NE)produced 26±5 secondary neurospheres. Thus, unlike EGF NSCs, which havethe capacity to passage into EGF or PDGF-AA, primary PRPs rarelyself-renew in either PDGF-AA or EGF.

To determine whether there were differences in the differentiation ofboth types of neurospheres, primary 6 DIV EGF- and PDGF-AA-generatedneurospheres were plated for 24 hours on poly-L-ornithine coatedcoverslips. Within 24 hours of plating, PDGF-AA-generated progenymigrated great distances (over 300 μm in some instances) from the centerof neurospheres (FIG. 2G). In contrast, primary EGF-generated progenyrarely migrated away from the center of differentiating neurospheres(FIG. 2H). Together these data demonstrate that PRPs are a populationdistinct from that of EGF-responsive NSCs.

Example 4

This example includes data indicating that BMP-2 and T3 promotedifferentiation of PRP into neurons and oligodendrocytes, respectively.

BMP and T3 direct astroglial and oligodendroglial differentiation ofO-2A progenitors, respectively (Ahlgren et al., Mol Cell Neurosci 9:420(1997); Mabie et al., J Neurosci 17:4112 (1997)). To determine whetherBMP-2 and T3 could direct differentiation of cells within PDGF-generatedneurospheres, six DIV primary PDGF-AA-generated neurospheres weredifferentiated on coverslips for 2 DIV, in 1% FBS, 50 ng/ml of BMP-2, 20ng/ml of T3, or T3 and BMP-2. Indirect immunocytochemistry revealed thatin the presence of 1% FBS, approximately 30% of the cells differentiatedinto β-III-tubulin expressing neurons, whereas 5% became O4 expressing;the remainder of the cells did not express either antigen (FIG. 3A andFIG. 3E). Compared to controls in 1% FBS (n=3; 23 NE), BMP-2 had nosignificant effect on the number (p>0.15; Tukey HSD; n=4; 38 NE) ofoligodendrocytes produced per clone (FIG. 3B). However, BMP-2 increasedneurite length in comparison to 1% FBS (FIG. 3B vs. 3A). In contrast, T3increased (5-fold) the differentiation of oligodendrocytes (p<0.001;Tukey HSD; n=4; 24 NE) (FIG. 3A vs. 3C; FIG. 3E). In the presence ofboth BMP-2 and T3, BMP-2 (n=4; 38 NE) suppressed T3-inducedoligodendrocyte differentiation of PDGF-generated progeny (p<0.001, T3vs. T3+BMP-2; Tukey HSD), and neuronal numbers were equivalent to thoseobserved in differentiation with BMP-2 alone (p>0.99; Tukey HSD) (FIG.3C vs. 3D; FIG. 3E). In all cases, numbers of differentiated cells(10-13 neurons and/or oligodendrocytes) remained constant atapproximately ⅓ of the total clone size (30-35%). GFAP-immunoreactivecells, indicative of astrocytes, were not detected in any of theseculture conditions.

Cells with an oligodendroglial morphology that expressed O4 and an innerring of β-III-tubulin were occasionally observed (FIG. 3F), but onlywhen differentiated in FBS. While these early neuronal andoligodendroglial antigens may not definitively identify bona fideneurons and oligodendrocytes, it is more likely that T3 and BMP-2 directthe fate choices of uncommitted PRPs. BMP-2 and T3 induction ofexpression of more mature neuronal and oligodendroglial antigens,respectively, in differentiating primary PDGF-generated neurospheres wasdetermined. PDGF-generated neurospheres differentiated for 2 DIVexpressed neurofilament M (NFM; neurons) or myelin basic protein (MBP;oligodendrocytes), but both antigens were never observed in the samecell, regardless of the differentiation conditions (FIG. 4A). BMP-2(n=3; 29 NE) increased the percentage of clones expressing NFM comparedto differentiation in 1% FBS (p<0.05; Tukey HSD; n=3; 22 NE), while T3(n=3; 26 NE) increased the number of MBP-only clones compared to 1% FBS,BMP-2 or T3 and BMP-2 (p<0.001; Tukey HSD; n=3; 27 NE) (FIG. 4B). Incontrast, BMP-2, when present with T3, completely inhibited thegeneration of MBP-only clones (p<0.001 T3 vs. T3+BMP-2; Tukey HSD) (FIG.4B). T3 (n=4; 29 NE) alone significantly increased the number ofMBP-expressing cells produced per clone compared to 1% FBS (n=4; 25 NE),BMP-2 (n=4; 27 NE), and T3 and BMP-2 (p<0.001 T3 vs. 1% FBS, BMP-2, andT3 and BMP-2; Tukey HSD; n=4; 28 NE) (FIG. 4C). These data indicate thatBMP-2 suppresses oligodendroglial differentiation but promotes neuronalmaturation, whereas T3 promotes the formation of oligodendrocytes fromPDGF-generated neurospheres.

Ventral forebrain PRPs therefore can generate neurons, and these neuronsarise from a common neuron-oligodendrocyte precursor that can be inducedto undergo neuronal differentiation with BMP-2, and oligodendroglialdifferentiation with thyroid hormone, triiodothyronine (T3). A commonneuron/oligodendrocyte precursor may exist in the developing forebrain.First, in vivo, a subset of PDGFRα-expressing cells co-express theTOAD-64 neuronal antigen. Second, PRPs give rise toparvalbumin-immunoreactive, GABAergic interneurons. Third, tangentialmigration of both oligodendrocytes and neurons is disrupted in Dlx1/2mutant mice, and BMP-2 enhances the generation of pure GABAergicneuronal clones at the expense of mixed neuronal/oligodendroglial clonesfrom premigratory stage MGE or LGE progenitors (Yung et al., Proc NailAcad Sci USA 99:16273 (2002)). Lastly, in vivo (Price et al.,Development 104:473 (1988); Grove et al., Development 117:553 (1993))and in vitro (Williams et al., Neuron 7:685 (1991)) retroviral lineagetracing studies of the E16 cortex have demonstrated the existence ofclones that could generate both neurons and white matter cells orneurons and oligodendrocytes, respectively, which have been suggested asthe cerebral equivalent of the O-2A progenitor. It is likely that thecells labelled were in fact PRPs that had migrated from the MGE to thecortex by E16, preliminary findings that PRPs are present in the E16cortex. Taken together, the data suggest a common precursor generatesoligodendrocytes and a subset of the interneurons in the forebrain, andthese results indicate that it is PRP.

Example 5

This example includes data indicating that BMP-2 with CNTF suppressesthe expression of OLIG2 and promotes astroglial differentiation. Theastroglial population is distinct from that of differentiated neuronsand oligodendrocytes.

GFAP-immunoreactive astrocytes were absent in PDGF-generatedneurospheres differentiated in BMP-2. Other investigators have reportedthat BMPs induce astrocyte differentiation of O-2A progenitors in vitro(Mabie et al., J. Neurosci 17:4112 (1997)) and glial progenitors in vivo(Gomes et al., Dev Biol 255:164 (2003)).

Additional studies were undertaken to ascertain the potential ofPDGF-generated progeny to differentiate into astrocytes in the presenceof CNTF, another factor known to induce astrocyte differentiation ofO-2A progenitors (Hughes et al., Nature 335:70 (1988)). CNTF on its owndid not induce GFAP expression in PDGF-generated progeny (FIG. 5A). Ithas been previously shown that LIF and BMP signaling can synergize toinduce astrocyte differentiation of fetal neural progenitors (Nakashimaet al., Science 284:479 (1999)). Studies on synergistic signaling mayalso reveal astrocyte differentiation in PDGF-generated neurospheres.When PDGF-generated progeny were differentiated in the presence of BMP-2and CNTF, a large number of GFAP-immunoreactive cells with astrocytemorphology were apparent (FIG. 5A). Indeed, PDGF-generated neurospheresdifferentiated into neurons and astrocytes, but not oligodendrocytes,with BMP-2 and CNTF (FIG. 5B). In addition, numbers of neurons thatdifferentiated in the presence of BMP-2 and CNTF (8±1; 24 NE) were notsignificantly different from the numbers observed with either BMP-2alone or BMP-2 and T3. In contrast, the numbers of undifferentiatedcells were dramatically reduced from 65-70% in FBS, BMP-2, T3 and BMP-2and T3 conditions to 2-4% (24 NE) in the presence of BMP-2 and CNTF.Thus, PRP progeny have the potential to differentiate into astrocytes.

The findings that the PRP progeny differentiate into astrocytes in thepresence of BMP-2 and CNTF suggest there may be a population of cellsdistinct from the N/O cells. If this were the case, then PDGF-generatedneurospheres treated with T3, followed by BMP-2 and CNTF should yieldclones that contain both oligodendrocytes and astrocytes.

Neurospheres (6 DIV) were differentiated in the presence of T3 for 3 DIVor in the presence of T3 with BMP-2 and CNTF added after the second day.Addition of BMP-2 and CNTF was delayed by two days to ensure theoligodendrocytes had been specified by the N/O cells and to preventtheir predominant differentiation into neurons by BMP-2. Neurospheresthat had BMP-2 and CNTF added to them after 2 DIV in T3 contained bothMBP-immunoreactive oligodendrocytes (4.9±0.7; n=3, 34 NE) andGFAP-immunoreactive astrocytes (2.4±0.4). In contrast, culturesdifferentiated in T3 contained oligodendrocytes (3.9±0.4), but noastrocytes (FIGS. 5C, D). Furthermore, the number of oligodendrocyteswere not reduced when BMP-2 and CNTF were added, indicating that BMP-2and CNTF do not promote differentiation of astrocytes from cells capableof oligodendrocyte differentiation.

It has been previously reported that BMP-2 overexpression in the chickspinal cord decreased expression of OLIG2 and oligodendrocytespecification (Mekki-Dauriac et al., Development 129:5117 (2002)). Morerecently, OLIG2 has also been found to directly suppress the astrocytedifferentiation pathway (Fukuda et al., Cell Death Differ 11:196(2004)).

To determine if OLIG2 expression is suppressed by BMP-2 or by BMP-2 andCNTF in PDGF-generated progeny, OLIG2 and O4 expression was studied byindirect immunocytochemistry after 6 DIV PDGF-generated neurospheres hadbeen differentiated for 2 DIV (FIG. 5E, F). BMP-2 significantly reducedexpression of OLIG2 in PDGF-generated progeny compared to MHM, 1% FBS,T3, T3+BMP-2, and CNTF. However, OLIG2 expression was still observed incultures differentiated in BMP-2, albeit in fewer cells and at arelatively reduced level. Although CNTF had no effect on OLIG2 or O4expression on its own, when combined with BMP-2, PDGF neurospheres lostvirtually all OLIG2 and O4 expression (FIGS. 5E, F). Loss of OLIG2expression alone cannot account for the induction of astroglialdifferentiation by BMP-2 and CNTF, considering that in the otherdifferentiation conditions astrocytes did not emerge even though asubstantial number of cells did not express OLIG2. These data indicatethat BMP-2 alone reduces OLIG2 expression, which may suppressoligodendrocyte differentiation and promote neuronal differentiation,whereas BMP-2 and CNTF together further reduce levels of OLIG2expression in PDGF-generated progeny, and promote astrocytedifferentiation in vitro.

Although not wishing to be bound to any theory, BMP-2 and CNTF maydepend on a complex of the transcription factors Stat3, Smad1, and theco-activators p300/CBP, which have been shown to induce astrocytedifferentiation of fetal neural progenitors (Nakashima et al., Science284:479 (1999)). A lack of such co-operative signaling may explainprevious observations that BMP signaling failed to promote astroglialdifferentiation (Wada et al., Dev Biol 227:42 (2000); Mekki-Dauriac etal., Development 129:5117 (2002)), although this may also be due toheterogeneity of OLP populations. The finding that BMP and CNTFsignaling co-operates in the differentiation of astrocytes correlateswith the repression of OLIG2 (and perhaps OLIG1) expression expandprevious observations that OLIG1/2 suppress astrocyte cell fatespecification (Zhou et al., Cell 109:61 (2002)) and glial fibrillaryacidic protein (GFAP) expression (Gabay et al., Neuron 40:485 (2003);Fukuda et al., Cell Death Differ 11:196 (2004)). Even if PRPs do notgenerate astrocytes during embryonic development, their contribution toglial scarring in injury has not been assessed, which leaves thepossibility that PRPs may generate astrocytes in vivo.

Example 6

This example includes data indicating that neurosphere generation byPDGF depends at least in part upon SHH signaling.

Signaling by SHH is necessary for the generation of OLPs in themammalian forebrain (Nery et al., Development 128:527 (2001);Tekki-Kessaris et al., Development 128:2545 (2001)). To determinewhether proliferation of PRPs may be sensitive to SHH signaling,dissociated MGEs (10,000 cells/ml) were grown in 100 ng/ml of PDGF-AAalone, or together with 5 μM cyclopamine, an inhibitor of SHH signaling(Cooper et al., Science 280:1603 (1998); Taipale et al., Nature 406:1005(2000)). Neurospheres generated in the presence of cyclopamine weresmaller than those generated in PDGF-AA+DMSO controls (compare FIG. 6Ato 6B). Cyclopamine also reduced the number of PDGF-AA-generatedneurospheres by 5-fold (p<0.003; t test; n=3) (FIG. 6C). There werelarge numbers of phase-bright differentiating cells in both conditions(indicated by arrows in FIG. 6B), indicating that the decrease inneurosphere numbers and size is likely not a result of a non-specifictoxic effect.

Since inhibition of SHH signaling attenuated the proliferation of PRPs,the effect of increasing SHH signaling on the number of neurospheresgenerated in the presence of PDGF was studies. Primary cells fromdissociated MGEs were cultured in the presence of PDGF-AA, 2 μg/ml ofthe 19-kDa amino-terminal fragment of SHH, or in the presence of bothfactors. FIG. 6D shows that, although SHH (n=3) had no effect on itsown, significantly more neurospheres (p<0.05; LSD test) were generatedin the presence of SHH and PDGF-AA (92±14; n=8) compared to PDGF-AA(60±9; n=8). Together, these data demonstrate that SHH is required inconcert with PDGF signaling for the proliferation and/or survival ofPRPs.

Example 7

This example includes data indicating that self-renewal of PRPs isdependent at least in part upon growth factor-dependent SHH signaling.

Initial studies suggested that PRPs lacked significant self-renewalcapacity (FIG. 2F). However, these studies employed a defiled culturemedia in which some factors required for self-renewal may be missing; Tofurther study PRP self-renewal, primary PDGF neurospheres from the E14MGEs of TgN(GFPU)5Nagy mice were grown for 7 DIV, individualneurospheres dissociated and transferred from single neurospheres ontopoly-L-omithine coated coverslips that had been plated with or without200,000 primary 7 DIV EGF-generated progeny 2 days earlier.

In the absence of the EGF-generated feeder layer or PDGF, large adherentcolonies of GFP-positive cells were rarely observed (FIG. 7A). Mostclones consisted of fewer than 10 cells (FIG. 7C). In contrast, singledissociates plated in the presence of PDGF and the EGF feeder layerproduced many clones of GFP-labeled cells that contained greater than 10cells (FIGS. 7B, C), and many cells expressed PDGFRα (FIG. 7D). Whensingle dissociates were plated on the EGF feeder layer but in theabsence of PDGF, few clones were observed and all were less than 10cells in number (FIG. 7C). These results suggest that some signalsinitiated by the EGF-generated feeder layer, whether soluble orcontact-dependent, contribute to self-renewal of PRPs together withPDGF.

Since SHH enhanced the number of primary neurospheres generated by PDGF(FIG. 4), the effect of SHH could on formation of secondary neurospheresfrom primary PDGF-generated neurospheres was studied. No significantdifference (p>0.99; Tukey HSD) in the number of secondary neurospheresgenerated when primary neurospheres were grown in the presence of PDGFand passaged into PDGF+SHH (n=3; 27 NE), or grown in the presence ofPDGF and SHH (n=4; 37 NE) and passaged into PDGF, compared to thosegrown and passaged in PDGF (n=7; 69 NE) (FIG. 7E): The results indicatethat self-renewal, as measured by the number of secondary neurospheres,cannot be augmented by SHH alone.

Signaling by FGF2, in combination with PDGF, has previously been shownto promote the self-renewal and inhibit the differentiation of O-2Aprogenitors in vitro, which normally lose responsiveness to PDGF afterseveral rounds of cell division and differentiate (Bogler et al., ProcNatl Acad Sci USA 87:6368 (1990)). A recent study reports that full SHHactions on oligodendrocyte development depends upon FGF2-stimulatedmitogen-activated protein kinase (MAPK) activity (Kessaris et al.,Development 131:1289 (2004)).

Precursors that expressed PDGFRα in the ventral forebrain were studiesfor expression of FGF receptors. FGF2 can bind to the four know FGFreceptors, FGFRs 1-4 (Reuss et al., Cell Tissue Res 313:139 (2003)).FGFR2 immunoreactivity was localized to nuclei in the ventricular zoneand to scattered cells within the MGE (FIG. 7F). Some of theFGFR2-labelled nuclei outside the ventricular zone clearly belonged tocells that expressed PDGFRα (arrows in FIG. 7F), indicating that bothsignaling pathways may regulate the proliferation and self-renewal ofPRPs.

To determine if FGF2 signaling alone, or together with SHH, augmentedthe generation of secondary neurospheres by PRPs, individual 6 DIVPDGF-generated neurospheres were dissociated and passaged in mediacontaining either FGF2 (n=7; 64 NE) or FGF2 and SHH (n=7; 60 NE). Noincrease in self-renewal was evident in PDGF-generated progeny that hadbeen passaged into FGF2 compared to PDGF (p>0.99; compare FIG. 7E to7G). However, in the presence of FGF2, SHH significantly enhancedself-renewal of PRPs (FIG. 7G), in comparison to primary PDGF-generatedneurospheres passaged into either FGF2 (p<0.05; Tukey HSD) or to PDGF(p>0.01; Tukey HSD; FIG. 7E).

The effect of PDGF and FGF2 in augmenting secondary neurosphereformation was studied. In the presence of PDGF+FGF2 (n=9; 78 NE), 27±3secondary neurospheres were generated (FIG. 7G), indicating that bothPDGF and FGF2 signaling contribute to self-renewal of PRPs, and this wasnot further increased when SHH was added (28±3 secondary neurospheres;p>0.99; Tukey HSD; n=5; 45 NE). The possibility that FGF2 is merelysupporting proliferation of PRPs is unlikely, given that primary PDGFneurosphere formation was unaffected by 1 μM SU5402 (an FGFR tyrosinekinase inhibitor) (Mohammadi et al., Science 276:955 (1997)), which wasable to block 90% of FGF2-induced NSC proliferation.

Despite the inability of added SHH to augment secondary PDGF neurosphereformation, co-operative actions of PDGF and FGF2 might be sufficient tosupport intrinsic SHH signaling, the latter of which is normallynecessary for oligodendrocyte generation. Indeed, self-renewal of PRPspassaged into PDGF and FGF2 was dependent on SHH signaling, ascyclopamine reduced the generation of secondary neurospheres (3±1; n=4;36 NE; FIG. 7G) to numbers closer to those obtained with PDGF (1±1) orFGF2 (3±1). Taken together, these findings indicate the full expressionof self-renewal capacity by PRPs is dependent, at least in part, onactivation of SHH signaling by both PDGF and FGF2.

PRPs have an extensive potential for expansion/self-renewal. Both PDGFand FGF2 were required for the formation of secondary neurospheres. Inaddition, PDGF and FGF signaling act through SHH to promote PRPself-renewal. Recent reports demonstrate that generation of OLPs by SHHis dependent on a basal level of MAPK activity, provided by FGFsignaling (Kessaris et al., Development 131:1289 (2004)). It is possiblethat MAPK plays a role in the regulation of SHH signaling in PRPself-renewal. It is noteworthy that the maximal number of secondary PRPs(26-28 neurospheres), derived from a primary PRP, is approximatelyequivalent to the number of undifferentiated cells within each PRPclone. Furthermore, undifferentiated cells are largely eliminated whenPRP clones are differentiated into neurons and astrocytes in thepresence of BMP-2+CNTF, although the neuron numbers are unchanged.

Taken together with the largely mutually exclusive differentiation ofneurons and oligodendrocytes in PRP clones, this leads to a proposedmodel for the lineage of PRPs (FIG. 8). As illustrated, PRPs are likelydescendents of multipotent NSCs, which is supported by findings thatthey can be generated by primary EGF-responsive NSCs and have identicalproperties to the PRPs from the ventral forebrain.

Example 8

This example includes data indicating that PRPs are responsive toneurotrophin-3 (NT-3).

The effect of brain-derived neurotrophic factor (BDNF), neurotrophin-3(NT-3), and nerve growth factor (NGF) on PRPs was determined. Cells werecultured as previously reported (Chojnacki et al., J Neurosci24(48):10888 2004) in the presence of various combinations of PDGF;brain-derived neurotrophic factor (BDNF), NT-3, nerve growth factor(NGF), and the effects on neurosphere generation characterized. MHM isthe defined medium used in cultures without growth factor.

As illustrated in FIG. 9, more neuroshperes are produced when PRPs aregenerated in the presence of PDGF and BDNF or NT-3, but not NGF. FIG. 10shows that NT-3 and BDNF promote the generation of larger neurospheresin the presence of PDGF. FIG. 11 shows that PRPs co-express PDGFRα andTrkC in the E14 ventral forebrain. FIG. 12 shows that PRPs do notco-express PDGFRα and TrkB in the E14 ventral forebrain

Dissociated primary cells were cultured in 24-well plates with orwithout NT-3 for 24 hours, stained for PDGFRα and Tunel, and the numberof labeled cells counted. As illustrated in FIG. 13, more PDGFRα-labeledcells were found in the NT-3 treated culture after 24 hours, but noTunel and PDGFRα co-labeled cells were observed in either condition. Theresults suggest that. NT-3 does not maintain the PRP population bypromoting cell survival. As illustrated in FIG. 14, an initial 24 hourtreatment with NT-3 was more effective at promoting the generation ofneurospheres than continued exposure to NT-3 after the first 24 hours.The data again suggests that NT-3 does not promote the survival of PRPs.

Seven day old primary neurospheres generated in either PDGF or PDGF+NT-3were dissociated and plated (25,000 cells/mL) in either PDGF or PDGF andFGF2. As illustrated in FIG. 15, neurospheres initially generated inPDGF and NT-3 produced more secondary neurospheres in either condition.The data indciates that NT-3 promotes self-renewal of PRPs.

PRPs are a unique population of oligodendrocyte precursors, with bothdistinct and similar properties to other OLPs described previously (Liuet al., Trends Neurosci 26:410 (2003); Noble et al., Dev Biol 265:33(2004); Rowitch, Nat Rev Neurosci 5:409 (2004)). The in vitro studieshave revealed that these precursors are heterogeneous in their abilityto generate neurons and subtypes of astrocytes and this is dependent onthe CNS region and developmental period of isolation. The earlydevelopment of hindbrain OLPs is unimpaired in OLIG2 null mice (Lu etal., Cell 109:75 (2002)), whereas there is a complete absence of OLPs inthe spinal cord, suggesting that OLPs in viva are also a heterogeneouspopulation. Even within the forebrain, we found that there may beheterogeneity in PRPs based on the expression of TOAD-64. Therefore, ifforebrain PRPs generate neurons in vivo, it may only be a subpopulationof PRPs that posses this capability. PRPs may maintain the capacity togenerate neurons through to adulthood. If human PRPs can be generated asneurospheres, this would permit isolating and expanding neuralprecursors for transplantation in white matter for the treatment ofinjury or disease.

1.-52. (canceled)
 53. An isolated clonally expanded or self-renewedpopulation of mammalian PDGF-responsive neural precursor (PRP) cellsthat express PDGF receptor alpha produced by a method comprisingculturing brain tissue in a culture medium containing PDGF underconditions allowing clonal proliferation or differentiation of the PRPcells.
 54. A method of increasing PRP cell numbers in a mammal,comprising administering a PDGFR agonist to the mammal in an effectiveamount for intracranial delivery of the PDGFR agonist to increase PRPcell numbers.
 55. The method of claim 54, wherein the PDGFR agonistcompromises PDGF.
 56. The method of claim 54, wherein the mammal doesnot receive EGF or FGF.
 57. The method of claim 54, further compromisingadministering FGF2, BDNF or NT-3 substantially simultaneously with thePDGFR agonist to the mammal.
 58. The method of claim 54, wherin thePDGFR agonist is administered to the brain of the mammal.
 59. The methodof any of claim 54, wherein the PDGFR agonist is administered locally,regionally, or systemically.
 60. The method of claim 54, wherein thePDGFR agonist is administered intracranially, intravenously,intravascularly, intramuscularly, subcutaneously, intraperitoneally,topically, orally, nasally or by inhalation.
 61. (canceled)
 62. A methodof producing oligodendrocytes, comprising: (a) culturing brain tissuefrom a mammal in a culture medium comprising a PDGFR agonist andallowing proliferation of PRP cells; and (b) differentiating theproliferated PRP cells to produce oligodendrocytes.
 63. The method ofclaim 62, wherein step (b) is performed by contacting the proliferatedPRP cells with an effective amount of thyroid hormone or T3.
 64. Themethod of claim 62, further compromising contacting the oligodendrocyteswith an effective amount of BMP-2 and CNTF to produce neurons andastrocytes.
 65. The method of claim 62, further compromising clonallyexpanding the proliferated PRP cells by contacting said cells with PDGFand FGF-2; or PDGF and BDNF; or PDGF and NT-3 prior to step (b).
 66. Amethod of producing neurons, comprising: (a) culturing brain tissue froma mammal in a culture medium comprising PDGFR agonist and allowingproliferation of PRP cells; and (b) differentiating the proliferated PRPcells to produce neurons.
 67. The method of claim 66, wherein step (b)is performed by contacting the proliferated PRP cells with an effectiveamount of BMP-2.
 68. The method of claim 66, further comprising clonallyexpanding the proliferated PRP cells by contacting said cells with PDGFand FGF-2; or PDGF and BDNF; or PDGF and NT-3 prior to step (b).
 69. Amethod of producing astrocytes, comprising: (a) culturing brain tissuefrom a mammal in a culture medium comprising PDGFR agonist and allowingproliferation of PRP cells; and (b) differentiating the proliferated PRPcells to produce astrocytes.
 70. The method of claim 69, wherein step(b) is performed by contacting the proliferated PRP cells with aneffective amount of BMP-2 and CNTF.
 71. The method of claim 69, furthercomprising expanding the proliferated PRP cells by contacting said cellswith PDGF and FGF-2; or PDGF and BDNF; or PDGF and NT-3 prior to step(b). 72.-76. (canceled)
 77. A method of increasing oligodendrocytes,neurons or astrocytes in a mammal, comprising: (a) administering aneffective amount of PDGFR agonist to the mammal to proliferate PRPcells; and (b) administering an effective amount of thyroid hormone orT3 to increase oligodendrocytes, BMP-2 to increase neurons, or bothBMP-2 and CNTF to increase astrocytes.
 78. The method of claim 77,further comprising administering FGF2, BDNF or NT-3 substantiallysimultaneously with the PDGFR agonist to the mammal.
 79. The method ofclaim 77, wherein the mammal is not administered EGF or FGF.
 80. Themethod of claim 77, wherein the PDGFR agonist, thyroid hormone, T3,BMP-2 or CNTF is delivered to the cranium of the mammal.
 81. The methodof claim 77, wherein the PDGFR agonist, thyroid hormone, T3, BMP-2 orCNTF is administered to the brain of the mammal.
 82. The method of claim77, wherein the PDGFR agonist is administered locally, regionally orsystemically.
 83. The method of claim 77, wherein the PDGFR agonist isadministered intracranially, intravenously, intravascularly,intramuscularly, subcutaneously, intraperitoneally, topically, orally,nasally or by inhalation.
 84. (canceled)
 85. The method of claim 77,wherein step (a) is performed prior to step (b).
 86. The method of claim77, wherein step (a) is performed at least one day prior, three daysprior or a week prior to step (b).
 87. The method of claim 77, whereinstep (a) is performed concurrently with step (b).
 88. The method ofclaim 77, wherein the mammal is in need of increased numbers ofoligodendrocytes, neurons or astrocytes.
 89. The method of claim 77,wherein the mammal suffers from a loss of or injury to oligodendrocytes,neurons or astrocytes.
 90. The method of claim 77, wherein the mammal isafflicted with or is at risk of affliction with a neurological diseaseor disorder, or undesirable medical condition.
 91. The method of claim90, wherein the neurological disease comprises a neurodegenerativedisease.
 92. The method of claim 90, wherein the neurological disease orundesirable medical condition comprises a stroke, aneurysm, brain orspinal cord injury or cranium or spinal column trauma.
 93. The method ofclaim 92, wherein the brain or spinal cord injury, or cranium or spinalcolumn trauma, is caused by a stroke or surgery.
 94. The method of claim93, wherein the stroke is hemorrhagic stroke, focal ischemic stroke orglobal ischemic stroke.
 95. The method of claim 90, wherein theneurological disease or undesirable medical condition affects central orperipheral nerves.
 96. The method of claim 95, wherein the centralnerves comprise brain or spinal cord.
 97. The method of claim 95,wherein the peripheral nerves comprise one or more of motor, sensory orautonomic nerves.
 98. (canceled)
 99. The method of treating orameliorating a disease, disorder or undesirable medical conditionassociated with neuron, oligodendrocytes or astrocyte loss, injury ordysfunction, comprising administering an effective amount of PDGFRagonist to a mammal harboring the disease, disorder or medicalcondition, as well as one or more of FGF-2, thyroid hormone, T3, BMP-2or CNTF.
 100. The method of claim 99, further comprising administeringto the mammal one or more agents selected from PDGF; PDGF and FGF-2;PDGF and BDNF; PDGF and NT-3; thyroid hormone; T3; BMP-2; BMP-2 andCNTF.
 101. The method of claim 99, wherein the undesirable medicalcondition comprises a neurological injury or trauma.
 102. The method ofclaim 101, wherein the neurological injury or trauma affects central orperipheral nerves.
 103. The method of claim 102, wherein the centralnerves comprise brain or spinal cord.
 104. The method of claim 102,wherein the peripheral nerves comprise one or more of motor, sensory orautonomic nerves.
 105. The method of claim 101, wherein the neurologicalinjury or trauma comprises stroke, aneurysm, brain or spinal cord injuryor cranium or spinal column trauma or injury.
 106. The method of claim105, wherein the stroke is hemorrhagic stroke, focal ischemic stroke orglobal ischemic stroke.
 107. The method of claim 99, wherein thedisease, disorder or undesirable medical condition comprises Alzheimer'sDisease, multiple sclerosis (MS), macular degeneration, glaucoma,diabetic retinopathy, peripheral neuropathy, Huntington's Disease,amyotrophic lateral sclerosis (ALS), Parkinson's Disease, stroke,depression, epilepsy, neurosis or psychosis.
 108. (canceled)
 109. Amethod of identifying an agent that modulates clonal proliferation orself renewal or differentiation of a neural precursor cell comprising:(a) providing an isolated or purified mammalian platelet derived growthfactor (PDGF)-responsive neural precursor (PRP) cell, wherein said cellexpresses PDGF receptor alpha, and wherein said cell, when contactedwith one or more of thyroid hormone, bone morphogenetic protein-2(BMP-2), ciliary neurotrophic factor (CNTF) or triiodothyronine (T3),gives rise to a differentiated neural cell that expresses detectableamounts of one or more protein markers selected from: GABA, parvalbumin,beta-II tubulin, calbindin D, calretinin, O4, neurofilament M (NFM),myelin basic protein (MBP), TOA-64/TUC-2 and GFAP or progeny cellsthereof; (b) contacting the cell or cells of step (a) with a candidateagent; and (c) determining if the candidate agent modulates clonalexpansion or differentiation of the cell or cells. 110-111. (canceled)112. A method of identifying an agent that modulates clonalproliferation or self renewal or differentiation of a neural precursorcell comprising: (a) providing an isolated or purified mammalianplatelet derived growth factor (PDGF)-responsive neural precursor (PRP)cell, wherein said cell expresses PDGF receptor alpha, and wherein saidcell, when contacted with one or more of thyroid hormone, BMP-2, CNTF orT3, gives rise to a differentiated neuron, oligodendrocyte, astrocyte ormixture thereof, or progeny cells thereof; (b) contacting the cell orcells of step (a) with a candidate agent; and (c) determining if thecandidate agent modulates clonal expansion or differentiation of thecell or cells.